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J. Biol. Chem., Vol. 281, Issue 21, 14729-14737, May 26, 2006

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Pleiotropic Effects of ATP·Mg²⁺ Binding in the Catalytic Cycle of Ubiquitin-activating Enzyme^*

Zeynep Tokgöz¹, Richard N. Bohnsack, and Arthur L. Haas²

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the Department of Biochemistry and Molecular Biology and the Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, December 21, 2005 , and in revised form, March 13, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP·Mg²⁺ binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp⁵⁷⁶ or Lys⁵²⁸ results in dramatically impaired binding affinities for ATP·Mg²⁺, a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k_cat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp⁵⁷⁶ and Lys⁵²⁸ are important for ATP·Mg²⁺ binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp⁵⁷⁶ within the activating enzymes for other ubiquitin-like proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The conjugation of ubiquitin and ubiquitin-like polypeptides to specific protein targets represents a fundamental and highly conserved strategy of eukaryotic cell regulation, reviewed most recently in Refs. 1–3. Post-translational modification by these polypeptides requires formation of isopeptide bonds through distinct yet evolutionarily related enzyme pathways that share a common mechanism in which the half-reactions of activation and ligation are catalyzed by distinct enzymes (1, 4). The ubiquitin-activating enzyme (Uba1)³ catalyzes the first step in the conjugation of ubiquitin to protein targets and serves as the archetype for paralogous steps in the activation of other Class 1 ubiquitin-like proteins, including Sumo (5, 6), Nedd8 (7, 8), ISG15 (9), Hub1 (10), FAT10 (11, 12), and Apg12 (13) among others, reviewed in Refs. 14 and 15. The reaction follows a mechanism in which binding of ATP·Mg²⁺ precedes binding of ubiquitin and the subsequent formation of a tightly bound ubiquitin adenylate intermediate (16, 17), a mixed acyl-phosphate anhydride between the carboxyl-terminal glycine of ubiquitin and AMP derived from the / cleavage of ATP (18). Ubiquitin adenylate is the immediate precursor for formation of a covalent Uba1-ubiquitin thiolester intermediate at a conserved active site cysteine (Cys⁶³², HsUba1a numbering), which is followed by a second round of ubiquitin adenylate formation to yield a Uba1 ternary complex composed of stoichiometric amounts of ubiquitin adenylate and ubiquitin thiolester (16). The Uba1-ubiquitin thiolester is the proximal donor of activated ubiquitin in formation of E2/Ubc-ubiquitin thiolesters required in all ubiquitin conjugation reactions (16, 19).

Because these conjugation pathways are universally conserved among eukaryotes⁴ but are absent from prokaryotes and Archaea (4), the evolutionary origins of ubiquitin ligation had remained an enduring paradox. However, recent studies reveal remarkable sequence similarities between the highly conserved E1⁵ paralogs and the MoeB subunit of Escherichia coli molybdopterin synthase, a multimeric enzyme complex that is involved in the evolutionarily conserved molybdenum cofactor (Moco) biosynthetic pathway found in both prokaryotes and eukaryotes (20, 21). The mechanism for molybdenum cofactor synthesis requires the MoeB-catalyzed activation of the carboxyl terminus of the 8757-Da MoaD subunit by formation of a tightly bound MoaD adenylate intermediate (20), a reaction mechanistically analogous to the Uba1-catalyzed activation of ubiquitin (16, 17). The 1.7-Å crystal structure of the MoeB-ATP-MoaD ternary complex of molybdopterin synthase provided early insights into the mechanism for the activation of MoaD, which shares a common -grasp fold and a carboxyl-terminal Gly-Gly motif with ubiquitin (21). Subsequent high resolution structures for free and substrate-bound forms of the human paralogs of the AppBp1-Uba3 Nedd8-activating enzyme (22, 23) and the Sae1-Sae2 Sumo-activating enzyme (24), in addition to parallel chemistries for carboxyl group activation of their cognate polypeptide substrates, reveal a common active site fold for this E1 superfamily and suggest that MoeB and MoaD are the previously unidentified evolutionary precursors for Uba1 and ubiquitin, respectively.

The active site residues of MoeB that interact directly with ATP are highly conserved among Uba1 orthologs and E1 paralogs for Sumo, Nedd8, and ISG15 (21, 22). Extant structures for this superfamily identify an aspartate residue that either directly (24) or inferentially (21, 23) interacts with Mg²⁺ chelated to bound ATP. The importance of this residue is suggested by its absolute conservation among MoeB and E1 paralogs as well as the dramatic effects of point mutagenesis. Mutation of Asp¹³⁰ to alanine abrogates MoeB activity in E. coli (21), whereas mutations of the paralogous Asp¹⁴⁶ of Uba3 or Asp¹¹⁷ of Sae2 significantly ablate Nedd8 and Sumo activation by human AppBp1-Uba3 or Sae1-Sae2 heterodimers, respectively (22, 24). However, loss-of-function mutations are rarely informative without additional studies, since the phenotype cannot be unambiguously interpreted. Thus, whereas the loss-of-function mutations that arise from nonconservative changes in Asp¹³⁰ and its paralogous residues are consistent with their predicted roles in ATP·Mg²⁺ binding, collateral mechanistic and/or structural effects cannot be precluded.

To understand more completely the mechanistic contribution of the aspartate residue in the catalytic cycle of human ubiquitin-activating enzyme and its paralogs, we have introduced a series of point mutations at Asp⁵⁷⁶ of human Uba1a that corresponds to Asp¹³⁰ of E. coli MoeB, Asp¹⁴⁶ of human Uba3, and Asp¹¹⁷ of human Sae2. Kinetic analyses confirm a role for Asp⁵⁷⁶ in ATP·Mg²⁺ binding. However, the point mutants also exhibit pleiotropic effects on the mechanism of ubiquitin activation, including transition state stabilization and order of substrate binding, that reveal a more complex role not anticipated in earlier studies. Such insights identify Asp⁵⁷⁶ and its paralogs as critical catalytic residues in the reaction cycle of this enzyme family.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine ubiquitin, creatine phosphokinase, and yeast inorganic pyrophosphatase were purchased from Sigma. The ubiquitin was further purified to apparent homogeneity and then radioiodinated by the chloramine-T procedure (25, 26). Carrier-free Na¹²⁵I, [2,8-³H]ATP, and Na₄³²PP_i were purchased from PerkinElmer Life Sciences. Recombinant human HsUbc2b and the active site mutant HsUbc2bC88A were those described previously (27).

Generation of GST-HsUba1a Point Mutants—A pGEX3X-E1 construct encoding the nuclear form of the human ubiquitin-activating enzyme (HsUba1a), generously provided by Dr. Alan L. Schwartz (Washington University School of Medicine), was used for the expression of wild type and mutant HsUba1a enzymes. The D576A, D576E, and D576N point mutants of HsUba1a were generated by the PCR overlap extension method of Ho et al. (28) using pGEX3X-E1 as a template. Point mutations were introduced using internal primers containing the desired mutation and external primers flanking the region of the gene bound by XhoI and Eco72RI restriction sites. Following PCR amplification of the corresponding single site mutations, inserts were subcloned into the pGEM-T vector. The subcloned insert was digested with XhoI/Eco72RI restriction enzymes, agarose gel-purified, and then ligated into a similarly restricted pGEX3X vector. Mutations were confirmed by sequencing the complete XhoI/Eco72RI inserts on an Applied Biosystems ABI Prism 3100 DNA sequencer.

Expression and Purification of GST-HsUba1a Wild Type and Mutant Proteins—E. coli BL21 (DE3) cells were transformed with pGEX3X encoding either wild type or mutant forms of ubiquitin-activating enzyme. Single colonies harboring the appropriate vector (pGEX3X-wtE1, pGEX3X-E1D576A, pGEX3X-E1D576N, or pGEX3X-E1D576E) were inoculated into 50 ml of LB medium containing 100 µg/ml ampicillin and then grown to stationary phase at 30 °C. This culture was used to inoculate 500 ml of LB-ampicillin medium at a final dilution of 1:25 and then grown to an A₆₀₀ of 0.6 at 30 °C with constant shaking. Protein expression was induced by the addition of isopropyl 1-thio--D-galactopyranoside to a final concentration of 0.3 mM, after which the culture was allowed to grow an additional 2 h. The cells were harvested by centrifugation and then resuspended in ice-cold 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 1 mM DTT. All subsequent steps were conducted at 4 °C. The resuspended cells were lysed by a French press, and the cell debris was removed by centrifugation at 100,000 x g for 30 min. The resulting supernatant was loaded onto a glutathione-agarose affinity column (Sigma) equilibrated with 50 mM Tris-HCl (pH 7.5) and 1 mM DTT. The GST wild type and mutant human Uba1a fusions were eluted with 20 mM reduced glutathione in 50 mM Tris-HCl (pH 7.5) and 1 mM DTT and then dialyzed overnight against 50 mM Tris-HCl (pH 7.5) containing 1 mM DTT. The purified proteins were analyzed by 7.5% SDS-PAGE, followed by Western blotting and subsequent visualization using anti-GST antibodies and ECL detection. Protein concentrations for full-length wild type and mutant HsUba1a enzymes were normalized to GST content using quantitative Western blotting.

Enzyme Assays—Concentrations of active wild type and mutant GST-HsUba1a enzymes were determined by the stoichiometric formation of ¹²⁵I-ubiquitin thiolester from free radioiodinated ubiquitin (5000 cpm/pmol) in end point assays, followed by nonreducing SDS-PAGE resolution and counting of associated ¹²⁵I radioactivity in excised bands (16). Enzyme-bound ubiquitin [³H]adenylate formation was determined using [2,8-³H]ATP (3.6 x 10⁴ cpm/pmol) and measured by trichloroacetic acid-precipitable radioactivity (16). Initial rates of ATP/³²PP_i exchange were measured at the indicated concentrations of ATP, ubiquitin, ³²PP_i, and either GST-HsUba1a or the corresponding Asp⁵⁷⁶ or Lys⁵²⁸ mutants (17). Initial rates of HsUbc2b-¹²⁵I-ubiquitin thiolester formation were measured by nonreducing SDS-PAGE in HsUba1a-catalyzed transthiolation assays at the indicated concentrations of ATP, ¹²⁵I-ubiquitin, and recombinant HsUbc2b (27). For Asp⁵⁷⁶ mutants of GST-HsUba1a, initial rates of HsUba1a-¹²⁵I-ubiquitin thiolester formation were measured directly by modification of the E2 transthiolation assay (27).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Only Full-length GST-HsUba1a Is Active—Expression of wild type or mutant GST-HsUba1a consistently yielded a mixture of full-length protein (M_r 136,000) and a ladder of GST-linked fusion protein fragments when detected by Coomassie staining after SDS-PAGE resolution (not shown) or by Western blot using anti-GST antibodies (Fig. 1). Although the protein expression yielded a mixture of full-length and partially degraded fusion protein, only full-length GST-HsUba1a was active in forming the corresponding ¹²⁵I-ubiquitin thiolester (Fig. 1). The amount of GST-HsUba1a-¹²⁵I-ubiquitin thiolester calculated from the specific radioactivity of the labeled polypeptide agreed well with that predicted from the protein content of the full-length GST-HsUba1a fusion protein estimated by Coomassie staining and comparison with a known amount of bovine serum albumin (not shown).

Thus, a carboxyl-terminal segment of 20 kDa, representing 170 amino acids, is essential for the activity of HsUba1. The cleavage point for this fragment lies between the predicted carboxyl-terminal -grasp domain common to all members of the E1 superfamily (22) and the thiolester active site located at Cys⁶³². However, other studies showed that truncation of the 118-residue predicted -grasp domain from the carboxyl terminus of HsUba1a had no effect on the stoichiometry of the resulting mutant⁶; therefore, the critical region must lie within a 52-amino acid segment between residues 888 and 940 of the intact protein. Crystal structures for MoeB, AppBp1-Uba3, and Sae1-Sae2 show that the paralogous regions for the latter critical segment comprise a buried helix and two runs of antiparallel sheet that constitute a contact face with their respective ubiquitin-like proteins. Therefore, truncation of this segment reasonably explains complete inactivation of the enzyme by abolishing binding of the -grasp polypeptide cosubstrate.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. SDS-PAGE resolution of recombinant GST-HsUba1a. Left lane, glutathione-Sepharose affinity-purified human GST-HsUba1a was resolved by SDS-PAGE on a 7.5% gel under reducing conditions and then electrophoretically transferred to polyvinylidene difluoride membrane and visualized by Western blotting using affinity-purified rabbit anti-GST antibody and ECL detection. The migration positions for full-length GST-HsUba1a and free GST are shown to the left, along with mobility markers of the indicated molecular weights. Right lane, 1 pmol of wild type GST-HsUba1a was incubated in an end point 125I-ubiquitin thiolester assay and then resolved in parallel by SDS-PAGE on the same gel as in the left lane but under nonreducing conditions (16). The GST-HsUba1a-125I-ubiquitin thiolester was visualized by autoradiography, the position of which is indicated to the right.

Initial velocity experiments with wild type GST-HsUba1a revealed strict hyperbolic kinetics based on the linearity of double reciprocal plots when ATP·Mg²⁺, ¹²⁵I-ubiquitin, or HsUbc2b was varied at saturating concentrations of the other cosubstrates (not shown). Values of K_m and k_cat, the latter defined as V_max/[GST-HsUba1a]_o, were calculated from nonlinear regression analyses of the respective data sets and are summarized in Table 2. In the presence of saturating ¹²⁵I-ubiquitin (5 µM) and recombinant HsUbc2b (0.5 µM), the K_m of 5.5 ± 0.7 µM for ATP was in good agreement with the value of 7.0 ± 1.1 µM reported for wild type human erythrocyte Uba1 (27). Likewise, when [¹²⁵I-ubiquitin]_o was varied at saturating ATP·Mg²⁺ (2 mM) and HsUbc2b (0.5 µM), wild type GST-HsUba1a exhibited a K_m of 0.8 ± 0.1 µM for radiolabeled ubiquitin that was identical to that previously determined for human erythrocyte Uba1 (27). Finally, the concentration dependence for the initial rate of HsUbc2b transthiolation versus [HsUbc2b]_o at saturating ATP·Mg²⁺ (2 mM) and ¹²⁵I-ubiquitin (5 µM) yielded a K_m of 111 ± 6nM that was in excellent agreement with the value of 123 ± 19 nM determined earlier for the human erythrocyte enzyme (27). The intrinsic k_cat for HsUbc2b transthiolation (k_cat,trans) corresponded to 3.4 ± 0.1 s^–1 when calculated from the V_max for GST-HsUba1a-catalyzed transthiolation in the latter experiment (Table 2) and agreed well with the value of 4.5 ± 0.3 s^–1 for erythrocyte HsUba1 (27).

Mutation of Asp⁵⁷⁶ Decreases the Rate of Ubiquitin [³H]Adenylate Formation—Earlier studies demonstrated that the stoichiometric formation of wild type Uba1 ternary complex is complete within the first 30 s of incubation with ATP·Mg²⁺ and ubiquitin at 37 °C (16, 27, 29). Fig. 2 illustrates that ternary complex formation catalyzed by recombinant wild type GST-HsUba1a is also rapid at 2 mM ATP·Mg²⁺ and 5 µM ¹²⁵I-ubiquitin when measured as the corresponding ¹²⁵I-ubiquitin thiolester (closed triangles). A parallel assay confirmed that ubiquitin [³H]adenylate formation was stoichiometric with GST-HsUba1a-¹²⁵I-ubiquitin thiolester at the earliest time point (30 s) in Fig. 2 (not shown), consistent with the results of Table 1 for the wild type recombinant enzyme. In contrast, ¹²⁵I-ubiquitin thiolester formation under identical conditions and with a nearly identical concentration of full-length GST-HsUba1aD576A, determined by end point ¹²⁵I-ubiquitin thiolester formation as described under "Materials and Methods," was markedly slower and required 20 min to reach completion (Fig. 2, closed circles). The time course for GST-HsUba1aD576A-¹²⁵I-ubiquitin thiolester formation followed strictly first order kinetics over at least six half-lives, based on the linearity of appropriate semilog plots (not shown), and yielded a pseudo-first order rate constant of 0.005 s^–1. In Fig. 2, the solid line through the data for GST-HsUba1aD576A (solid circles) represents a theoretical first order fit for k_o = 0.005 s^–1. The linearity of such first order plots precludes significant contributions from catalytically active degradation fragments having radically different kinetics, in agreement with conclusions drawn from the wild type stoichiometries of the recombinant enzyme preparations. Results qualitatively similar to those of Fig. 2 were obtained with the GST-HsUba1aD576E and GST-HsUba1aD576N mutants and yielded pseudo-first order rate constants of 0.003 and 0.006 s^–1, respectively (not shown). These observations demonstrate that mutation at Asp⁵⁷⁶ results in a 10³-fold reduction in the apparent first order rate constant for GST-HsUba1a-¹²⁵I-ubiquitin thiolester formation compared with the lower limit of 5 s^–1 estimated previously for the intrinsic rate constant for ubiquitin [³H]adenylate formation catalyzed by wild type enzyme (27, 31).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Time course for GST-HsUba1aD576A-125I-ubiquitin thiolester formation. The time course for 125I-ubiquitin thiolester formation with 1.5 pmol (30 nM) of wild type GST-HsUba1a (closed triangles) per datum point or an equivalent amount of full-length GST-HsUba1aD576A (closed circles), determined by Coomassie staining following SDS-PAGE as in Fig. 1, was determined at 37 °C in 50-µl incubations containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 10 mM MgCl2, 1 mM DTT, 1 mg/ml carrier bovine serum albumin, and 5 µM 125I-ubiquitin (16, 17). Aliquots taken at the indicated times were quenched in 50 µl of SDS sample buffer without -mercaptoethanol and then resolved by nonreducing 7.5% SDS-PAGE and visualized by autoradiography. The absolute content of 125I-ubiquitin thiolester was quantitated by -counting the excised bands for each time point and calculated using the specific activity of the radiolabeled polypeptide (16, 17). The line through the data for GST-HsUba1aD576A represents a theoretical first order fit for ko = 0.005 s–1.

In contrast, the end point stoichiometries for the ubiquitin [³H]adenylate intermediate formed with each of the three mutants were only 1% of their respective thiolester values (Table 1). We have previously found that mutation of specific ubiquitin residues promotes dissociation of the otherwise tightly enzyme-bound ubiquitin [³H]adenylate and its subsequent hydrolysis by contaminating ubiquitin carboxyl-terminal hydrolases (31). In the present case, the apparent substoichiometric ubiquitin [³H]adenylate formation did not arise by similar dissociation of the intermediate from the enzyme and cleavage by any contaminating hydrolases, since exogenous free ubiquitin [³H]adenylate prepared by trichloroacetic acid precipitation of wild type Uba1 ternary complex (16, 18) was stable when added to the recombinant GST-HsUba1a mutant preparations (not shown). Previous kinetic studies have demonstrated that formation of enzyme-bound ubiquitin adenylate is rapid and that the subsequent transfer to form the covalent Uba1-ubiquitin thiolester is the rate-limiting step of ternary complex formation with the wild type enzyme (16, 17). This accounts for the constant 1:1 stoichiometry of thiolester/adenylate with time (16, 17). That ubiquitin [³H]adenylate is undetectable during the first 5 min of the time course in Fig. 2 and remains at only 1% of the end point thiolester for the three Asp⁵⁷⁶ mutants requires that under the conditions of the assay, ubiquitin [³H]adenylate formation must be rate-limiting and that the mutations have altered the equilibrium constant for formation of this intermediate.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Hyperbolic dependence for the initial velocity of GST-HsUba1aD576A-125I-ubiquitin thiolester formation on ATP concentration. Initial rates of GST-HsUba1aD576A-125I-ubiquitin thiolester formation were determined at 37 °C in 1-min incubations of 25-µl total volume containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mg/ml carrier bovine serum albumin, 10µM 125I-ubiquitin, 52 nM GST-HsUba1aD576A, and the indicated concentrations of ATP as described previously (16, 27). A hyperbolic concentration dependence with respect to ATP concentration was confirmed by linearity of the corresponding double reciprocal plot. Relevant kinetic constants were determined by nonlinear regression (27).

Since Asp⁵⁷⁶ functions in ATP·Mg²⁺ binding, confirmed by the results of Fig. 3 and Table 2, we did not a priori expect to observe an effect of the Asp⁵⁷⁶ mutations on ubiquitin binding. However, mutation of Asp⁵⁷⁶ to alanine significantly reduced the affinity of the point mutant for ¹²⁵I-ubiquitin (Fig. 4B) relative to that exhibited by wild type GST-HsUba1a (Fig. 4A) and yielded a K_m of 29 ± 6 µM (Table 2) when measured by the initial rate of GST-HsUba1aD576A-¹²⁵I-ubiquitin thiolester formation at saturating ATP (2 mM). The V_max from Fig. 4B yielded a k_cat of 0.024 ± 0.003 s^–1, which must represent the intrinsic first order rate constant for ¹²⁵I-ubiquitin adenylate formation (k_cat,AMP-Ub) (Table 2). In contrast, mutation of Asp⁵⁷⁶ to asparagine increased the K_m for ubiquitin binding to GST-HsUba1aD576N only 5-fold (K_m = 4.0 ± 0.5 µM) and yielded a corresponding k_cat,AMP-Ub of 0.011 ± 0.001 s^–1, Table 2. Although the GST-HsUba1aD576E point mutant showed no significant effect on the binding of ¹²⁵I-ubiquitin when measured as K_m, the relatively conservative point mutation exhibited the greatest effect on k_cat,AMP-Ub for ubiquitin adenylate formation (0.005 ± 0.001 s^–1) (Table 2), suggesting that the increase in side chain length for glutamate has a substantial effect on formation of the transition state for enzyme-bound ubiquitin adenylate. The results of Table 2 indicate that mutation of Asp⁵⁷⁶ has a much less dramatic effect on the binding of ubiquitin within the HsUba1a adenylate active site than found for binding of ATP·Mg²⁺ and that this effect is qualitatively proportional to the extent to which the mutations ablate ATP·Mg²⁺ binding. In contrast, the ability of HsUba1a to catalyze ubiquitin adenylate formation is markedly sensitive to mutation at Asp⁵⁷⁶, implicating this residue in transition state stabilization of this step.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Hyperbolic 125I-ubiquitin concentration dependence for wild type and Asp576 mutant GST-HsUba1a. A, initial rates of wild type GST-HsUba1a-catalyzed HsUbc2b-125I-ubiquitin thiolester formation were determined in 30-s incubations under the conditions described in the legend to Fig. 3 except that [ATP]o was held at 2 mM and 125I-ubiquitin was present at the indicated concentrations. B, initial rates for GST-HsUba1aD576A-125I-ubiquitin thiolester formation were determined in 1-min incubations identical to those described in the legend to Fig. 3, except that [ATP]o was held constant at 2 mM and 125I-ubiquitin was present at the indicated concentrations. Lines through the data represent hyperbolic nonlinear regression fits from which the relevant constants summarized in Table 2 were determined.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Dependence of initial velocity on HsUbc2b concentration for GST-HsUba1aD576A-catalyzed HsUbc2b transthiolation. Initial rates of GST-HsUba1aD576A-catalyzed HsUbc2b-125I-ubiquitin thiolester formation were determined in 25-µl incubations at 37 °C for 10 min in assays containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 10 mM MgCl2, 1 mM DTT, 1 mg/ml carrier bovine serum albumin, 2.5 nM GST-HsUba1aD576A, 10 µM 125I-ubiquitin, and the indicated concentrations of HsUbc2b (27). Hyperbolic binding kinetics were confirmed by the linearity of the corresponding double reciprocal plot. Values for Km and Vmax values were calculated by nonlinear hyperbolic regression analysis and are summarized in Table 2.

The three Asp⁵⁷⁶ point mutants exhibited K_m values for HsUbc2b binding that were in good agreement with that found for wild type GST-HsUba1a (Table 2), indicating that this residue does not contribute either directly or indirectly to HsUbc2b binding. The corresponding apparent k_cat values for HsUbc2b transthiolation were calculated from V_max/[GST-HsUba1a mutant]_o. We anticipated that the intrinsic k_cat for HsUbc2b transthiolation (k_cat,trans), when corrected for saturating [ATP]_o and [ubiquitin]_o, would correspond to the intrinsic k_cat,AMP-Ub values determined for mutant-bound ubiquitin adenylate formation, since the latter represented the rate-limiting step for ternary complex formation and, therefore, the rate-limiting step for subsequent HsUbc2b-¹²⁵I-ubiquitin thiolester formation. However, the three mutants exhibited intrinsic k_cat,trans values that were statistically greater than k_{cat, AMP-Ub} (Table 2). In the presence of GST-HsUba1aD576A, k_cat,trans for HsUbc2b transthiolation (0.120 ± 0.010 s^–1) was 5-fold greater than the k_{cat, AMP-Ub} for the corresponding ubiquitin adenylate formation. The GST-HsUba1aD576E and GST-HsUba1aD576N mutants also catalyzed greater intrinsic rates of HsUbc2b transthiolation than predicted from their corresponding values of k_{cat, AMP-Ub} for ubiquitin adenylate formation (Table 2). The k_cat,trans for the D576E mutant (0.100 ± 0.010 s^–1), was 20-fold greater than k_{cat, AMP-Ub} (0.005 ± 0.001 s^–1), whereas the D576N mutant exhibited a 2-fold greater k_cat,trans (Table 2). These differences suggest that HsUbc2b transthiolation enhances the rate of ubiquitin adenylate formation. This effect is not due to allosteric activation by HsUbc2b binding alone, since substitution of 0.5 µM HsUbc2bC88A, which is incapable of forming a thiolester (27), did not result in a measurable increase in k_cat,AMP-Ub with GST-HsUba1aD576A (not shown). Therefore, the stimulation in observed k_cat requires the active site Cys⁸⁸ of HsUbc2b.

Mutation of Asp⁵⁷⁶ Alters the Mechanism of Co-substrate Binding for GST-HsUba1a—The mechanism of ubiquitin activation by rabbit reticulocyte Uba1 exhibits absolutely ordered binding in which ATP·Mg²⁺ is the leading and ubiquitin the trailing substrate (17). Ordered binding typically implies the presence of an obligatory conformational change upon binding of the leading substrate that allows the subsequent binding of the trailing substrate. Paradoxically, the observation that selected ubiquitin point mutants support random addition mechanisms with wild type Uba1 indicates that ordered substrate addition and the presumed conformational change are not requisites for the catalytic cycle of ubiquitin activation (31). This conclusion is supported by our recent finding that the catalytic cycle of Nedd8 activation by heterodimeric AppBp1-Uba3 follows a pseudo-ordered but formally random mechanism in which ATP·Mg²⁺ is the preferred leading substrate, although Nedd8 is capable of binding first at high concentrations (29). The effects of the Asp⁵⁷⁶ mutations on the binding affinities for ATP and ubiquitin suggested that the mechanism of substrate addition might also be affected. To test this prediction, ATP/³²PP_i isotope exchange kinetics were used to examine the order of substrate addition among wild type GST-HsUba1a and the three Asp⁵⁷⁶ point mutants.

Wild type GST-HsUba1a and the Asp⁵⁷⁶ point mutants exhibited hyperbolic kinetics for their dependence of the initial rate for ATP/³²PP_i exchange on [ATP]_o (data not shown). When the ubiquitin concentration was varied at a constant ATP concentration (2 mM), wild type GST-HsUba1a showed hyperbolic behavior below 2 µM ubiquitin, indicated by the linearity of the corresponding double reciprocal plot (not shown), and yielded an estimated K of 0.17 ± 0.01 µM and an extrapolated V_max of 65 ± 1 pmol/min (k_cat = 6.0 ± 0.1 s^–1). However, substrate inhibition was observed at higher ubiquitin concentrations that tended to a limiting initial rate of 5 pmol/min, representing 8% of the extrapolated V_max (Fig. 6A). This indicates that human Uba1a exhibits a pseudo-ordered mechanism for substrate binding, with ATP·Mg²⁺ being the preferred leading substrate and ubiquitin the preferred trailing substrate (17). In contrast, hyperbolic kinetics were observed at all ubiquitin concentrations tested for GST-HsUba1aD576A (Fig. 6B) and GST-HsUba1aD576E (Fig. 6C), demonstrating that the point mutations shift the enzyme to a purely random addition mechanism for substrate binding. The D576A mutant had the most dramatic effect on the isotope exchange kinetics, shifting the concentration dependence to near linearity, from which we could only estimate a lower limit to the K of 5 ± 2 mM and a k_cat of 0.7 ± 0.9 s^–1.⁷ Although K cannot be directly equated with the corresponding K_m value, as has been discussed previously (31), the marked increase in K for GST-HsUba1aD576A is consistent with the significantly larger K_m for ATP (Table 2). The effect of the D576E mutant was much less severe and yielded a K of 283 ± 12 µM and a k_cat of 1.3 ± 0.1 s^–1. We cannot preclude substrate inhibition for the Asp⁵⁷⁶ mutants at higher ubiquitin concentrations than those tested in Fig. 6, B and C; however, since substrate inhibition is apparent within 2-fold of the K for wild type GST-HsUba1a, it unlikely that the point mutants would show similar substrate inhibition at still higher concentrations. These results indicate that coordination of Asp⁵⁷⁶ with the metal of ATP·Mg²⁺ is coupled to events in the adenylate active site responsible for pseudo-ordered substrate addition, providing a mechanistic insight not apparent from the extant crystal structures for this enzyme family.

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Dependence of ubiquitin concentration on the initial rate of GST-HsUba1a-catalyzed ATP/PPi exchange. Initial velocities of ATP/32PPi exchange were determined at 37 °C in 50-µl incubations containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 10 mM MgCl2, 0.1 mM 32PPi, 1 mM DTT, the indicated concentrations of ubiquitin, and either GST-HsUba1a or Asp576 mutant (17). A, ubiquitin dependence for wild type GST-HsUba1a (3.6 nM); B, ubiquitin dependence for GST-HsUba1aD576A (3.2 nM); C, ubiquitin dependence for GST-HsUba1aD576E (4 nM). The solid lines represent nonlinear hyperbolic regression fits.

The kinetics of GST-HsUba1aK528A-¹²⁵I-ubiquitin thiolester formation were determined in parallel to those of the Asp⁵⁷⁶ point mutants, the results of which are summarized in Table 2. Mutation of Lys⁵²⁸ lowers the affinity for ATP·Mg²⁺ somewhat less than mutating Asp⁵⁷⁶ to alanine but consistent with the predicted contribution of the Lys⁵²⁸ hydrogen bond to the -phosphoryl oxygen of ATP·Mg²⁺. The Lys⁵²⁸ mutation also slightly alters the affinity for ubiquitin (Table 2). Like the Asp⁵⁷⁶ mutants, GST-HsUba1aK528A exhibits a wild type affinity for HsUbc2b (Table 2). Finally, in ATP/PP_i isotope exchange assays similar to those of Fig. 6, GST-HsUba1aK528A exhibited purely hyperbolic kinetics with respect to [ubiquitin]_o, indicating random substrate addition (not shown). These results are consistent with the kinetic phenotypes of the Asp⁵⁷⁶ and Lys⁵²⁸ mutants arising by generalized effect on ATP·Mg²⁺ binding.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present studies demonstrate for the first time that alterations in the ATP·Mg²⁺ binding site of Uba1 exhibit a remarkably pleiotropic phenotype in the activation of ubiquitin that could not have been anticipated from the crystal structures currently available for members of the E1 superfamily. Lake et al. (21) originally proposed that Asp¹³⁰ of MoeB contributed to ATP binding by interacting with the chelated Mg²⁺, supported by the loss-of-function phenotype in nitrate reductase overlay assays when this group was mutated to alanine. Paralogous mutation of Asp¹⁴⁶ within the Uba3 subunit of human Nedd8-activating enzyme supported this functional role, since it abrogated ³²P-labeled Nedd8 adenylate formation and subsequent thiolester formation at the active site Cys²¹⁶ of Uba3 (22). Similar observations have been made following mutation of Asp¹¹⁷ of Sae2 within the human Sae1-Sae2 heterodimeric activating enzyme for Sumo (24). Finally, the structure for ATP·Mg²⁺ bound to SAE1-SAE2 is consistent with coordination of the conserved aspartate with the chelated metal (24). Collectively, these results suggest a central role for this residue in the mechanism of these enzymes, presumably reconciled by its predicted contribution to ATP·Mg²⁺ binding. However, given the high affinity with which members of the E1 superfamily bind ATP·Mg²⁺ (17, 27, 29), the observed loss-of-function phenotypes for these mutations require abrogation of ATP·Mg²⁺ binding, which is difficult to reconcile with additional interactions between the nucleotide and its binding site. In the present paper, we quantitatively demonstrate the contribution of Asp⁵⁷⁶ of human Uba1 to the affinity of ATP·Mg²⁺ binding within the adenylate active site; more importantly, the data show that Asp⁵⁷⁶ is critical to downstream events in the catalytic cycle of ubiquitin activation that instead account for the loss-of-function phenotype.

Wild type human Uba1a has a marked affinity for ATP (K_m = 5.5 ± 0.7 µM), corresponding to a G_binding of 7.2 kcal/mol, presumably ensuring that the rate of ubiquitin activation is independent of fluctuations in this cosubstrate (4). The affinity of human Uba1 for ATP is reduced 38-fold by mutation of Asp⁵⁷⁶ to alanine (K_m = 208 ± 15 µM) (Table 2), an effect that is consistent with the proposed role of this residue in coordinating with the chelated metal of ATP·Mg²⁺ (21, 24) but insufficient to produce a loss-of-function phenotype at millimolar intracellular nucleotide concentrations (32). The increase in K_m for ATP binding between the wild type and D576A mutant corresponds to a G_binding contribution of 2.1 kcal/mol for Asp⁵⁷⁶, much less than the total binding energy of the nucleotide.⁸ The residual binding affinity of the Asp⁵⁷⁶ point mutant requires additional contributions from other active site interactions. The Lys⁵²⁸--phosphoryl hydrogen bond inferred from crystal structures (21, 23, 24) accounts for one of these interactions, since mutation of this residue to alanine results in a K_m of 119 ± 17 mM (Table 2), representing a G_binding of 1.8 kcal/mol. By comparison, Bacillus stearothermophilus tyrosyl-tRNA synthetase catalyzes a biochemically analogous ATP-coupled carboxyl group activation during the formation of a high energy tyrosyl adenylate intermediate. Lysine 82 of the enzyme hydrogen-bonds to the -phosphoryl oxygen of ATP·Mg²⁺, and mutation of this group to alanine yields a G_binding of 1.3 kcal/mol (33). Therefore, the binding contribution of Lys⁵²⁸ within the HsUba1a nucleotide binding site is consistent with related active site interactions.

Much smaller effects on ATP·Mg²⁺ binding affinity of 5-fold, corresponding G_binding values of 0.8 or 1.0 kcal, are observed when Asp⁵⁷⁶ is mutated to either glutamate or asparagine, respectively (Table 2). The reduced binding of GST-HsUba1aD576E probably arises from steric constraints imposed by the longer side chain of glutamate in its interaction with bound ATP·Mg²⁺. The G_binding of 1.0 kcal/mol for GST-Uba1aD576N approximates a value of 1.7 kcal/mol for the difference in GTP·Mg²⁺ binding within the G domain of EF-Tu following mutation of Asp⁸⁰, a residue that similarly binds the Mg²⁺ of the nucleotide chelate (34). That asparagine substitutes remarkably well for Asp⁵⁷⁶ with respect to ATP·Mg²⁺ binding suggests that interaction of the latter with chelated Mg²⁺ is not due to Coulombic contributions. Coordination of asparaginyl and glutaminyl side chains to Mg²⁺ have been observed previously for wild type enzymes, such as bacterial glutathione synthetase, D-alanine, D-alanine ligase, and pyruvate phosphate dikinase, among others (35, 36).

Several lines of evidence demonstrate that mutating Asp⁵⁷⁶ alters the rate-limiting step for generating the HsUba1a ternary complex from the step for formation of the HsUba1a-ubiquitin thiolester to that of ubiquitin adenylate (Tables 1 and 2). For wild type ubiquitin-activating enzyme, generation of the ternary complex containing both HsUba1a-ubiquitin thiolester and its tightly bound ubiquitin adenylate precursor is rapid with respect to E2 transthiolation (27, 29). However, mutation of Asp⁵⁷⁶ significantly reduces the rate of ubiquitin adenylate formation and allows the time course for HsUba1a ternary complex formation to be observed directly (Fig. 2). That the effect of Asp⁵⁷⁶ mutation is at the step of ubiquitin adenylate formation and not the subsequent step of HsUba1a-ubiquitin thiolester formation is supported by the marked change in end point stoichiometry for wild type versus mutant ternary complex with the three substitutions tested (Table 1).

The kinetic assays are unable directly to resolve the intrinsic rate constant for k_cat,AMP-Ub on the step for ubiquitin adenylate formation catalyzed by wild type Uba1 (17). However, a lower limit for wild type k_cat,AMP-Ub of 6.0 ± 0.1 s^–1 can be estimated from the extrapolated k_cat for ATP/PP_i isotope exchange at saturating ATP·Mg²⁺ and ubiquitin (Fig. 6A), which agrees well with the lower limit of 9.6 s^–1 determined for rabbit Uba1 (17).⁹ Using the former limiting value, mutation of Asp⁵⁷⁶ to alanine results in a >250-fold decrease in k_cat,AMP-Ub relative to wild type enzyme (Table 2). The overall effect of mutating Asp⁵⁷⁶ can be quantitatively compared by considering the catalytic specificity of HsUba1a with respect to ATP·Mg²⁺, defined as k_cat,AMP-Ub/K_m,ATP. For wild type GST-HsUba1a, the lower limit for k_cat,AMP-Ub/K_m,ATP can be estimated as 1.1 x 10⁶ M s^–1, whereas the corresponding value for GST-HsUba1aD576A is 115 M s^–1. The consequence of the mutating Asp⁵⁷⁶ to alanine thus represents a >9600-fold decrease in the overall efficiency of ubiquitin adenylate formation, an effect that renders HsUba1a functionally inactive under all but the most sensitive assay conditions. This suggests that the loss-of-function phenotype observed for the paralogous mutation in other members of this family results from a combination of reduced affinity and compromised catalytic efficiency (21, 23, 24). Reduction in k_{cat, AMP-Ub} also manifests as a significantly diminished equilibrium constant for ubiquitin adenylate formation, normally 0.1 for wild type enzyme (17, 37), since it constitutes the numerator of this term (17), altering the end point stoichiometry for the resulting HsUba1a ternary complex (Table 1).

We could not have anticipated a priori that mutation of Asp⁵⁷⁶ or Lys⁵²⁸ would so dramatically lower k_cat,AMP-Ub and the stoichiometry for the final ternary complex. Aspartate 576 is unlikely to serve an overt catalytic role as a general acid-base group, since glutamate does not effectively substitute for Asp⁵⁷⁶ with respect to the stoichiometry of the ternary complex or the magnitude of k_cat,AMP-Ub (Tables 1 and 2). A functional ternary complex of correct stoichiometry can be reconstituted by the addition of exogenous ubiquitin adenylate to wild type Uba1 (18), even when enzyme and ubiquitin adenylate are both treated with 20 mM EDTA; therefore, we can preclude an alternative model for Asp⁵⁷⁶ in which Mg²⁺ remains associated with this residue to facilitate nucleophilic attack of Cys⁶³² during subsequent Uba1-ubiquitin thiolester formation. However, because the phenotype of Lys⁵²⁸ mutation is similar to that observed with the three Asp⁵⁷⁶ mutants, it is likely that they all derive from a common mechanism.

Aminoacyl tRNA synthetases catalyze a carboxyl group activation that is identical in chemistry to the reaction of Uba1 and other E1 paralogs (16, 38). These enzymes catalyze a highly conserved in line nucleophilic attack of the substrate carboxyl group on the -phosphate of ATP·Mg²⁺ to form the corresponding bound aminoacyl adenylate and PP_i·Mg²⁺ (38–40). The reaction passes through a trigonal bipyramide pentacovalent phosphoryl transition state in which the relative positions of the oxygen and phosphorous atoms are proposed to shift little from their ground states (38, 39). Catalysis in this enzyme class arises in large part from enhanced binding and concomitant stabilization of the incipient transition state (38, 41, 42). Thus, mutation of active site groups involved in substrate binding that are also critical for binding and stabilizing the transition state invariably leads to marked reductions in k_cat (42, 43). As a corollary, mutation of groups not involved in initial substrate binding but that are required for transition state stabilization also shows significant k_cat effects (42, 43).

For HsUba1a, the significant decrease in k_cat,AMP-Ub requires, by definition, effect that the of mutating Asp⁵⁷⁶ and Lys⁵²⁸ groups in the ATP·Mg²⁺ binding site results in destabilization of the transition state. Thus, the data of Table 2 suggest that mutation of Asp⁵⁷⁶ destabilizes the transition state by 1.2 kcal/mol for HsUba1aD576A, 2.1 kcal/mol for HsUba1aD576E, and 1.4 kcal/mol for HsUba1aK528A (Table 2). The latter energy of destabilization agrees favorably with a value for G of 1.7 kcal/mol for mutation of Lys⁸² to alanine in tyrosyl-tRNA synthetase noted earlier (33). Therefore, the common chemistries of these otherwise unrelated enzyme classes appear to manifest a conserved catalytic mechanism of transition state stabilization.

Ternary complex formation is normally too rapid to follow by the manual assays used in the present studies, necessitating our use of E2 transthiolation as a coupled reporter assay (27). Since mutation of Asp⁵⁷⁶ or Lys⁵²⁸ inhibits formation of bound ubiquitin adenylate, we were able to monitor subsequent HsUba1a-¹²⁵I-ubiquitin thiolester formation directly (Fig. 2). Thus, V_max determined under these conditions and extrapolated to saturating substrate reflects the intrinsic value of k_cat,AMP-Ub for the respective single point mutants (Table 2). Because kinetics measure only the rate-limiting reaction of a multistep pathway, in this case ubiquitin adenylate formation, we were surprised to find that k_cat,trans values independently determined by HsUbc2b transthiolation failed to agree with those determined directly by HsUba1a-¹²⁵I-ubiquitin thiolester formation (Table 2). Observation that the dominant negative mutant HsUbc2bC88A failed to enhance k_cat,AMP-Ub rules out models in which the E2 acts as a positive allosteric effector of bound ubiquitin adenylate formation; however, the observation requires that stimulation by HsUbc2b requires Cys⁸⁸. Earlier studies by Pickart et al. (44) demonstrated that occupancy of the nucleotide/adenylate site by either ATP·Mg²⁺ or ubiquitin adenylate enhanced the rate of E2 transthiolation by 13-fold. Microscopic reversibility requires that occupancy of the E2 binding site must also stimulate bound ubiquitin adenylate formation. Since HsUbc2bC88A fails to stimulate ubiquitin adenylate formation, the data require that the observed difference between k_cat,AMP-Ub and k_cat,trans (Table 2) reflects a positive allosteric effect of bound wild type HsUbc2b on the rate of ubiquitin adenylate formation and that this effect is observed only upon binding of wild type uncharged E2.

The inhibition pattern for the ubiquitin dependence of ATP/PP_i exchange demonstrates that wild type human Uba1 exhibits pseudoordered substrate addition (Fig. 6A) (17). The pseudo-ordered addition mechanism results from the combined effects of differences in binding site geometry and differential relative affinities for ATP·Mg²⁺ versus ubiquitin as the leading substrate, requiring that wild type human Uba1 possess a greater affinity for ATP·Mg²⁺ as leading substrate than for ubiquitin. Mutation of Asp⁵⁷⁶ to either alanine or glutamate shifts GST-HsUba1a to a random addition mechanism, revealed by the hyperbolic ATP/PP_i exchange kinetics with varying ubiquitin concentration (Fig. 6, B and C). The K_m values for ATP versus ubiquitin differ too little among the Asp⁵⁷⁶ mutants to allow us to propose that the change in binding order results from differential affinities (Table 2). More likely, Asp⁵⁷⁶ coordination to Mg²⁺ triggers subsequent ubiquitin binding, possibly by opening the channel through which the carboxyl terminus of the latter threads, as revealed from crystal structures for the adenylate binding sites of AppBp1-Uba3 and Sae2-Sae3 (22–24).

The present studies demonstrate that Asp⁵⁷⁶ is a critical residue in the catalytic cycle of ubiquitin-activating enzyme for ATP·Mg²⁺ binding, substrate binding order, and transition state stabilization that go beyond its originally proposed role based on structural information. However, the complex kinetic phenotype arising from mutation of this group is explained fully by its contributions to substrate and transition state binding. The present data also reveal a previously unrecognized coupling between ATP·Mg²⁺ and ubiquitin binding as well as coupling between E2 binding and the rate of ubiquitin adenylate formation.

	FOOTNOTES

 
^* This work was supported by United States Public Health Service Grant GM34009 (to A. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: St. Jude Children's Research Hospital, Dept. of Structural Biology, 332 N. Lauderdale, Memphis, TN 38105-2794.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-3004; Fax: 504-568-3370; E-mail: ahaas{at}lsuhsc.edu.

³ The abbreviations used are: Uba1, ubiquitin-activating enzyme; DTT, dithiothreitol; E1, generic term for activating enzymes of Class 1 ubiquitin-like proteins; E2/Ubc, ubiquitin carrier protein or ubiquitin-conjugating enzyme; HsUba1a, human nuclear form of ubiquitin-activating enzyme; HsUbc2b, "b" isoform of the human/rabbit ortholog of Saccharomyces cerevisiae Rad6/Ubc2 (also termed E2_14Kb); GST, glutathione S-transferase; Ub, ubiquitin.

⁴ The single exception is the ubiquitin-like protein ISG15, which is found only among vertebrates and thus represents an evolutionarily recent diverged pathway apparently required for unique functions (45, 46).

⁵ We shall use E1 generically to refer to the paralogous activating enzymes for Class I ubiquitin-like proteins.

⁶ Z. Tokgöz and A. L. Haas, unpublished observation.

⁷ The atypically large S.E. values for these kinetic constants reflect the problem associated with fitting a hyperbolic curve over a limited range near or below the inflection point (17).

⁸ Binding energy estimates calculated from the corresponding G_binding values are not strictly additive due to entropic contributions.

⁹ Since ubiquitin adenylate formation is not rate-limiting for wild type E1 ternary complex formation, it is not technically possible directly to determine k_cat,AMP-Ub. However, a lower limit can be estimated from the k_cat for ATP/PP_i exchange, since the latter assays are measured at equilibrium (17).

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Alan L. Schwartz for kindly providing the pGEX3X-E1 expression plasmid and to Adam Harder for technical support.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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