The 2′-Phosphate of NADP Is Responsible for Proper Orientation of the Nicotinamide Ring in the Oxidative Decarboxylation Reaction Catalyzed by Sheep Liver 6-Phosphogluconate Dehydrogenase*

Sheep liver 6-phosphogluconate dehydrogenase shows a high specificity for NADP, with a much lower affinity for NAD. Discrimination between NADP and NAD suggests that the interactions between the 2′-phosphate and 6-phosphogluconate dehydrogenase contribute most of the binding energy for NADP. There are three active site residues, Asn-32, Arg-33, and Thr-34, that hydrogen-bond to the 2′-phosphate of NADP according to the crystal structure of the E·Nbr8ADP complex. In this study alanine mutagenesis was used to probe the contribution of each of the three residues to binding the cofactor and to catalysis. All mutant enzymes exhibit no significant change in V/Et or K6PG but an increase in KNADP that ranges from 6- to 80-fold. All mutant enzymes also exhibit at least a 7-fold increase in the primary kinetic 13C-isotope effect-1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Asn-32, Arg-33, and Thr-34 in providing binding energy for NADP, and more importantly, the 2′-phosphate of NADP is required for proper orientation of the cofactor to allow rotation about the N-glycosidic bond as it is reduced in the hydride transfer step.

6-Phosphogluconate dehydrogenase (6PGDH 2 ; EC 1.1.1.44) catalyzes the reversible oxidative decarboxylation of 6-phosphogluconate (6PG), producing ribulose-5-phosphate and CO 2 with the concomitant reduction of NADP to NADPH. The kinetic mechanism is rapid equilibrium random on the basis of a complete kinetic characterization of the enzymes from sheep liver and Candida utilis (1,2). The pH dependence of kinetic parameters indicates a general acid-general base chemical mechanism (1,2), and site-directed mutagenesis studies suggest that Lys-183 and Glu-190 are likely the general base and the general acid, respectively (3,4). In this mechanism the general base (Lys-183) is required to accept a proton from the 3-hydroxyl group of 6PG concomitant with hydride transfer from C-3 of 6PG to the coenzyme. Reduction of the nicotinamide ring is accompanied by rotating around the N-glycosidic bond such that the ring occupies the position formerly occupied by the 1-carboxylate of the substrate (5). The resulting 3-keto-6phosphogluconate intermediate is decarboxylated to produce the enediol of ribulose-5-phosphate with the general base used to protonate the carbonyl oxygen. A general acid (Glu-190) is needed to facilitate the tautomerization of the enediol of ribulose-5-phosphate to the keto product (Fig. 1).
6PGDH is specific for the substrate 6PG; the best alternative substrate is 6-phosphomannonate, which has a V/E t 10 3 -fold lower and a V/K 6PM E t 10 5 -fold lower than values obtained with 6PG (6). The enzyme also has a very high specificity toward its cofactor NADP; the V/K NAD E t is 10 5 -fold lower than that obtained with NADP (7). Most of the direct interactions between the protein and the cofactor are made to the 2Ј-phosphate (8). Removal of the 2Ј-phosphate of NADP decreases the affinity more than 1000-fold (7). In the structure of the E⅐Nbr 8 ADP (the active NADP analog) binary complex, the 2Ј-phosphate interacts with three active site residues that are within hydrogen-bond distance: Asn-32, Arg-33, and Thr-34 (6). Multiple sequence alignment of 6PGDH shows that Asn-32 is completely conserved, whereas Arg-33 is replaced by tyrosine in the Bacillus licheniformis and Bacillus subtilis sequences, which can use NAD as a cofactor, and Thr-34 is replaced by serine in some species (Fig. 2). Data indicate that identical hydrogen-bond potential can be retained at residues 32 and 34, whereas the ionic interaction at residue 33 is likely important for binding the 2Ј-phosphate.
At least four hydrogen bonds have been proposed between the 2Ј-phosphate and the three residues discussed above, two of which are contributed by Arg-33; Fig. 3 shows these residues in the protein environment. Mutation of Arg-34 in the 6PGDH from Lactococcus lactis (the homolog is Arg-33 in sheep liver 6PGDH) to tyrosine gave a mutant enzyme that lost most of its affinity for NADP (700-fold decrease) and used NAD as a cofactor but very poorly (9). In addition to the three residues discussed above, a helix dipole (␣b) is positioned 5 Å away from the 2Ј-phosphate and is proposed to provide an additional electrostatic interaction with the 2Ј-phosphate (8).
In this paper we investigate the role of each of the residues that interact with the 2Ј-phosphate of NADP. Site-directed mutagenesis was used to change Asn-32, Arg-33, and Thr-34 to Ala, one at a time. Initial velocity and isotope effects studies were carried out to characterize the mutant enzymes. Data are consistent with the hypothesis that these residues are important in providing the binding energy for the 2Ј-phosphate of NADP. They also play a role in catalysis in the 6PGDH reaction as a result of properly orientating the cofactor as well as 6PG.

Chemicals, Reagents, Bacterial
Strains, and Plasmids-Oligonucleotide primers, chemicals, and reagents were obtained from sources described previously (10). The XL1-Blue strain of Escherichia coli was used to transform the mutated plasmid, and M15[pREP4] was the host strain for expression of the mutant proteins.
Site-directed Mutagenesis-Mutagenesis was carried out using the QuikChange site-directed mutagenesis. Double-stranded DNA prepared from recombinant plasmid pPGDH.LC4 (11) was used as a template, and the synthetic oligonucleotide primers are listed in Table 1. Whole gene sequencing was performed for every mutation at the Laboratory for Genomics and Bioinformatics of the University of Oklahoma Health Science Center in Oklahoma City. The resulting sequence was compared with that of the wild type 6PGDH using BLAST. Successfully  mutated plasmids were transformed into M15[pREP14] competent cells, the expression host. Frozen stocks of strains harboring plasmid were stored in LB/ampicillin/kanamycin medium containing 15% glycerol at Ϫ80°C.
Growth and Purification Conditions-Bacterial growth, expression, and purification of the mutant enzymes was as described previously (10). Protein concentrations were measured for all fractions using the method of Bradford (12). The wild type and mutant proteins were purified in an identical manner, and all enzymes were stored at 4°C in the same buffer used for elution from the ADP-agarose column.
Initial Velocity Studies-Initial rates were measured with a Beckman DU640 UV/visible spectrophotometer monitoring the appearance of NADPH (⑀ 340 ϭ 6.22 mM Ϫ1 cm Ϫ1 ). Temperature was maintained at 25°C using a Beckman temperature controller. For all enzymes, the initial rate was measured as a function of 6PG (15-500 M) at different fixed levels of NADP (1-3000 M) dependent on the mutant enzyme. All assays were measured in 100 mM Hepes, pH 7.5. Initial velocity studies were also performed with NAD as the cofactor at a saturating concentration of 6PG (40 K m ).
Primary Deuterium Isotope Effects-Concentrations of 3-h-6PG and 3-d-6PG were determined enzymatically in triplicate via endpoint assay using wild type 6PGDH. The concentrations from three determinations were in agreement within 2%. Primary deuterium isotope effects, D V and D (V/K 6PG ), were obtained by direct comparison of initial velocities in triplicate varying 3-h-6PG or 3-d-6PG at saturating concentrations of NADP (14).
pH Studies-The pH dependence of V and V/K NADP was measured at saturating 6PG (40 K m ). The pH was maintained using the following buffers at 100 mM concentrations: Bis-Tris, 5-6.5; Hepes, 6.5-8.5; Ches, 8.5-9.5. Sufficient overlap was obtained upon changing buffers to eliminate buffer effects. The pH was determined before and after the initial velocity measurements. 13 C Kinetic Isotope Effect-Isotope effects were measured as described previously (10) using the natural abundance of 13 C at the C-1 position of 6PG (15). High conversion (100% reaction, which represents 12 C/ 13 C in the substrate 6PG) and low conversion samples were used to measure the 12 C/ 13 C isotope ratios in the CO 2 produced from the reaction of 3-h-6PG or 3-d-6PG (16). From these ratios, the 13 C kinetic isotope effect was calculated (17).
Isotopic composition of the CO 2 was measured on a Finnigan Delta E isotope-ratio mass spectrometer in the laboratory of Dr. Michael Engel, Department of Geophysics, University of Oklahoma. All ratios were corrected for 17 O according to Craig (18).
Data Processing-Double reciprocal plots of the data were visually evaluated, and all plots and replots were linear. Data were fitted using the appropriate rate equations and programs developed by Cleland (19). Data for substrate saturation curves obtained at a fixed concentration of the second substrate were fitted using Equation 1. Initial velocity patterns were fitted using Equation 2. Deuterium kinetic isotope effect data were fitted using Equation 3. In Equations 1 and 2, v is the initial velocity, V is the maximum velocity, A and B are reactant concentrations, K a and K b are the Michaelis constants for NADP and 6PG, respectively, and K ia is the dissociation constant for E⅐NADP. In Equation 3, F i is the fraction of deuterium label in FIGURE 3. Residues involved in the binding site of 2-phosphate of NADP in sheep liver 6-phosphogluconate dehydrogenase. C is green, N is blue, O is red, and P is magenta. The dashed lines represent potential hydrogen bonds between the residues of interest and the cofactor. The numbers above the dashed lines are the distances in Å between the hydrogen bond donors. The figure was created using PyMOL from DeLano Scientific LLC. The accession number in the Protein Data Bank is 1PGN for the E⅐Nbr 8 ADP structure.

TABLE 1 Sequence of oligonucleotide primers
Subscripts f and r represent forward and reverse primers. Mutated codons are in bold.
the substrate, and E V and E V/K are the isotope effects Ϫ1 on V and V/K. The pH dependence of V/K was fitted using Equation 4, whereas that for V was fitted using Equation 5. In Equations 4 and 5, y is V or V/K, C is the pH independent value of y, H is the hydrogen ion concentration, and K 1 and K 2 are the acid dissociation constants for enzyme or substrate groups important in a given protonation state for optimal binding and/or catalysis.
Calculation of 13 C kinetic isotope effects was performed according to Equation 6, where f is the fraction of completion of the reaction, and R p and R ∞ are the 12 C/ 13 C isotopic ratios for CO 2 at partial and complete conversion, respectively. Isotope ratios, given as ␦ 13 C, were calculated from Equation 7, where R smp and R std are 12 C/ 13 C isotopic ratios for sample and standard, respectively. The standard for CO 2 was calibrated from Pee Dee Belemnite with a 12 C/ 13 C of 0.0112372 (18).

RESULTS
Spectral Properties of Mutant Enzymes-Far UV CD spectra were recorded for all mutant and wild type enzymes, and all were identical after adjusting for protein concentration (data not shown). In addition, identical tryptophan fluorescence emission spectra were obtained for all mutant and wild type enzymes upon excitation at 298 nm, indicating that the microenvironment of tryptophan residues of the proteins is the same for all enzymes (data not shown). There are 16 tryptophan residues in 6PGDH spread throughout the protein structure. As a result, there are no major changes in the overall structure of the enzyme resulting from the mutation, and changes are restricted to the local area within the active site.
Kinetic Parameters of the Mutant Enzymes-Initial velocity patterns were obtained by measuring the initial rate at pH 7.5 using variable concentrations of 6PG (15-500 M) and NADP (1-1000 M) dependent on the mutant enzyme. Data are summarized in Table 2. No significant change was observed in K 6PG or in the V/E t for all three mutant enzymes with respect to the values of the wild type enzyme. K NADP increased from 6-to 80-fold for all mutations, resulting in a 6 -80-fold decrease in V/K NADP E t . Data indicate elimination of either asparagine or arginine impairs cofactor binding more than it does when the threonine is replaced.
With NAD as the cofactor, K NAD increases by more than 1000-fold compared with K NADP ; in fact saturation by NAD cannot be achieved, and only V/K NAD E t can be estimated. For the wild type enzyme, a value of 1.1 ϫ 10 Ϫ7 M Ϫ1 s Ϫ1 was obtained for V/K NAD E t , whereas for the N32A, R33A, and T34A mutant enzymes, values of 5 ϫ 10 Ϫ7 M Ϫ1 s Ϫ1 , 5 ϫ 10 Ϫ9 M Ϫ1 s Ϫ1 , and 3.7 ϫ 10 Ϫ8 M Ϫ1 s Ϫ1 , respectively, were estimated. Data suggest that substitution of the three residues by alanine do not improve the ability of the enzyme to use NAD as a cofactor.
Kinetic Primary Deuterium Isotope Effects-The kinetic isotope effects on V and V/K 6PG were measured at saturating NADP (40 K m ) ( Table 3). The isotope effects are smaller than the values obtained with the wild type enzyme in all cases and within error equal to each other, consistent with the proposed rapid equilibrium random kinetic mechanism (1). 13 C Kinetic Isotope Effects-Data for 13 C kinetic isotope effects obtained with 3-h-6PG and 3-d-6PG are shown in Table  3. For all of the mutant enzymes, the value of 13 (V/K 6PG ) H Ϫ1 is at least 7-fold higher than that of the wild type protein. Deuteration of 6PG decreases the observed 13 C kinetic isotope effects in all cases, and the equality for a stepwise mechanism with oxidation preceding decarboxylation is satisfied within error (16).
pH Dependence of Kinetic Parameters-The pH dependence of the kinetic parameters was measured by varying NADP at saturating levels of 6PG (40 K m ). All of the mutant and the wild type enzymes are stable from pH 5.5 to 9.5. For all of the mutant enzymes, a bell shaped pH-rate profile with limiting slopes of 1 and Ϫ1 was obtained for V/K 6PG . The pH-rate profile for V is bell-shaped in the case of the N32A and R33A mutant enzymes, whereas that of the wild type and T34A mutant enzymes decreases only at low pH. The pK a values are summarized in Table 3. The pH-rate profiles for the wild type and R33A mutant enzyme are shown in Fig. 4 and Fig. 5.

DISCUSSION
The main aim of this research was to determine the importance of residues Asn-32, Arg-33, and Thr-34, which interact with the 2Ј-phosphate of NADP, and their roles in providing binding energy. Site-directed mutagenesis was used to change Asn-32, Arg-33, and Thr-34 to alanine one at a time to eliminate the interaction between each of the residues and the 2Ј-phosphate of NADP. Steady-state kinetic parameters and isotope effects were measured to determine the effect of the substitutions on the ability of 6PGDH to use NADP as a cofactor.
Theory for interpretation of kinetic parameters and isotope effects in the 6PGDH reaction has been developed previously, and equations are reproduced here for aid in data interpretation (5,13). The oxidative decarboxylation reaction catalyzed by 6PGDH is stepwise with oxidation preceding decarboxylation as suggested by multiple deuterium/ 13 C kinetic isotope effect studies (13). Multiple solvent deuterium/ 13 C kinetic isotope effect and proton inventory studies indicate the presence of an isomerization of the enzyme complex before hydride transfer and decarboxylation (20). The kinetic mechanism of 6PGDH can be written, (Eq. 8) where A and B represent NADP and 6PG, respectively, and X, Q, and R represent 3-keto-6PG, ribulose 5-phosphate, and   NADPH, respectively. The rate constants k 3 and k 4 are for binding and dissociation of 6PG, and k 3Ј and k 4Ј are for binding and dissociation of NADP, k 5 and k 6 are for an isomerization of E⅐NADP⅐6PG complex, k 7 and k 8 are for forward and reverse hydride transfer, and k 9 is the rate constant for decarboxylation of the 3-keto intermediate and release of CO 2 . Given the rapid equilibrium nature of the mechanism, central complex interconversion is rate-limiting (steps included from EAB to EЈQR) and, thus, k 4 and k 4Ј , Ͼk 5 . The following expressions have been developed.
(Eq. 13) and c r ϭ k 8 k 9 (Eq. 14) A kinetic deuterium isotope effect is observed on the hydride transfer step, depicted by D k 7 and D k 8 , which can be related by the equilibrium isotope effect, D K eq ϭ D k 7 / D k 8 . Expressions for the primary kinetic deuterium isotope effects are given in Equations 15 and 16.
Because D V ϭ D (V/K) (Table 3), c f ϭ c Vf , and the equation for the isotope effect on V (Equation 15) is equal to that on V/K, i.e. Equation 16. The rate constant k 9 will reflect a 13 C kinetic isotope effect given by 13 k 9 . Expressions for the primary kinetic 13 C kinetic isotope and the multiple 13 C/D multiple isotope effect, i.e. the 13 C kinetic isotope effect with 3-d-6PG, are given in Equations 17 and 18. Commitment factors assume the hydride transfer step and not the decarboxylation step is isotope-sensitive.
where D k 7 is the intrinsic deuterium isotope effect on the hydride transfer step, and D K eq is the equilibrium isotope effect on hydride transfer (1.18 for oxidation of a secondary alcohol (21)). K 6PG is the ratio of V and V/K 6PG , and K NADP is the ratio of V and V/K NADP and taking into account c f ϭ c vf , which requires k 9 ϾϾ k 5 and k 6 ϾϾ k 5 .
Therefore, K 6PG is the equilibrium constant for dissociation of 6PG from E⅐NADP⅐6PG, and K NADP is the equilibrium constant for dissociation of NADP from E⅐NADP⅐6PG. Using the above rate equations, we discuss the results for each of the mutant enzyme.
The key to interpretation of the data obtained for the mutant enzymes lies in changes in K NADP , D (V/K 6PG ) and 13 (V/K 6PG ). The decrease in K NADP likely results from an increase in the net off-rate for NADP from E⅐NADP⅐6PG.

Kinetic Parameters
A significant increase in K NADP has been obtained for all three mutant enzymes. The positively charged arginine side chain is thought to help to neutralize the charge on the 2Ј-phosphate; it is also responsible for all the contacts to one face of the adenine ring and, thus, forms one side of its binding pocket, shielding it from solvent (8). Changing arginine to alanine not surprisingly decreases the affinity of the mutant enzyme for NADP by Ͼ70-fold. A similar phenomenon has been observed for glutathione reductase (22), indicating that this interaction may be one of the solutions to the problem of determining the specificity of the enzyme for NADP.
In the case of Asn-32, the amide side chain donates a hydrogen bond to the 2Ј-phosphate, and it also interacts with the 3Ј-hydroxyl of the adenine ribose. The amide of Asn-32 bridges two turns between ␤A-␣a and ␤B-␣b (Fig. 3) and is also within hydrogen-bonding distance of the main chain nitrogen of Leu-10 and the main chain carbonyl of Thr-34, forming a hydrogen-bond network (8). Substitution of asparagine with alanine eliminates all of the above interactions, and thus, a 60-fold decrease in the affinity to NADP is not unexpected. Similar binding energy is provided by residues Asn-32 and Arg-33 (2.4 kcal/mol and 2.5 kcal/mol, respectively). T34A exhibits the smallest change in K NADP , indicating this residue does not contribute as much as Asn-32 and Arg-33 in providing the binding energy to NADP. If a simple sum of ⌬⌬G o values could accurately be used to give the overall ⌬G o of NADP binding, the three residues together contribute about 78% of the total binding energy to NADP, with about 68% derived from Asn-32 and Arg-33. Although the simple sum is not valid, data are consistent with significant roles of Asn-32, Arg-33, and Thr-34 in providing the binding energy of NADP.

pH Studies
The pH dependence of the kinetic parameters measured for the wild type enzyme in this study is slightly different from the data reported previously. The maximum velocity exhibits a single pK of 6.4, whereas two pK values of 5.8 and 8.8 were suggested in an earlier study, although the decrease at high pH was slight (1,3). The difference may reflect the difference in stability of the enzyme after ADP-agarose affinity chromatography, a step not carried out in the earlier study.
In the V pH-rate profile of the N32A and R33A mutant enzymes, the pK value of Lys-183 is shifted to a lower pH, 6 -6.2, compared with 6.4 for the WT enzyme. The V/K pHrate profile of all the three mutant enzymes exhibits two pK values, one on the acid side and one on the basic side, as observed for the WT enzyme (3). However, the pK values are closer together in the case of the mutant enzymes, largely a result of an increase in the observed pK of the group with the pK on the acid side of the profile. The pK values are attributed to Glu-190 (acid side) and Lys-183 (basic side), which serve as general acid and general base, respectively; that is, the two groups exist in reverse protonation states. It is suggested that the local environment of the general acid and the general base in the mutant enzymes is more hydrophobic than they are in the wild type enzyme. It is difficult to explain the change in the hydrophilicity of the local environment unless the structures of the mutant enzymes are available.

Kinetic Isotope Effects
Qualitative Analysis-A decrease in D V and D (V/K) was obtained, whereas 13 (V/K 6PG ) H Ϫ1 is increased about 6-fold compared with the WT enzyme (Table 3). Data suggest an increase in the reverse rate of the hydride transfer step and a decrease in the rate of the decarboxylation step. Using Equations 15-17, to have decreased D V and D (V/K) and an increased 13 (V/K), the reverse commitment to catalysis c r (k 8 /k 9 ) has to increase; that is, the partition ratio of the 3-keto-6-phosphogluconate changes to favor 6PG. Given the increase in c r and in order that V for the mutant enzymes remain unchanged compared with that of the WT enzyme, another rate process must increase to compensate. The obvious choice is k 7 since k 7 /k 8 determines the equilibrium constant for the hydride transfer step, which is unlikely to change as a result of any of the mutations.
Quantitative Analysis-A quantitative analysis of the data supports the above conclusions and extends the interpretation of the isotope effects. The largest primary 13 C kinetic isotope effects were observed for enzymes with mutations in the 6-phosphate site, and the largest of these was 1.0397, measured for the K260A mutant enzyme (10). Pre-steady-state kinetic studies of the K260A mutant enzyme give a full stoichiometric burst of NADPH production, and thus, 13 k Ϸ 1.04. A primary deuterium kinetic isotope effect of 2.5 was observed for the M13I mutation in 6PGDH (5). In addition, a quantitative analysis of multiple 13 C/D isotope effects for the wild type enzyme published previously (13) allowed estimates of the value of commitment factors and intrinsic isotope effects. For values of 13 k from 1.025-1.1, the value of D k varied over a narrow range, 2.8 -3.4, with a value of about 3 estimated for a 13 k value of 1.04. Using these estimates of 13 k and D k, estimates of c f , c r , and c Vf should be obtained that satisfy Equations 15-17 and generate the observed isotope effects for all three mutant enzymes, assuming the transition states for hydride transfer and decarboxylation do not change significantly. Because D V ϭ D (V/K), c f ϭ c Vf ; thus, only c f will be discussed. Values of the parameters of interest are given in Table 4, and all satisfy Equations 15-17. As can be seen from Table 4, the reverse commitment does indeed increase consistent with partitioning of the 3-keto-6PG intermediate in favor of 6PG. In addition, c f Յ c r ; a value of almost 4 is obtained for c f /c r for the WT enzyme, whereas values range from 0.4 to 1 for the mutant enzymes. Overall, this results in a decrease in the commitment for the primary 13 C kinetic isotope effect, (1 ϩ c f )/c r from 6 for WT to 0.9 -1.7 for the mutant FIGURE 6. Free energy diagram for WT and mutant 6PGDHs. Energy levels of the intermediates are arbitrary, whereas those for the conformational change (EAB to EЈAB) and the decarboxylation step (EЈXR to EЈQR) are relative to the hydride transfer step (EЈAB to EЈXR). The solid line represents WT enzyme, whereas 1, 2, and 3 represent the profiles for N32A, R33A, and T34A, respectively. enzymes. This can be shown in terms of a free energy diagram (Fig. 6). Note that for each of the mutant enzymes the barrier, relative to hydride transfer, decreases for the conformational change and increases for decarboxylation. However, the overall barrier height for the reaction does not change significantly as required given the lack of change in V/E t ( Table 2). In fact, the highest barrier is slightly lower than that for the WT enzyme in the case of the R33A and T34A mutant enzymes, in agreement with their slightly higher V/E t values. Thus, it is the change in c f /c r , not just the increase in c r that generates the large increase in 13 (V/K) with a smaller decrease in D (V/K). The change in c f is almost certainly attenuated as a result of an increase in k 7 as suggested above.

Conclusions
We have recently provided evidence that reduction of the nicotinamide is accompanied by rotation of the ring about the N-glycosidic bond to a position that was occupied by the 1-carboxylate of 6PG (Fig. 1). The end result would be elimination of the hydrogen-bonding interaction between Glu-190 and the 1-carboxylate, facilitating decarboxylation and resulting in the re face of the nicotinamide ring being exposed to the active site (the stereochemistry of hydride transfer is to the si face). It is likely that changing the interaction with the 2Ј-phosphate by eliminating the side chain of Asn-32, Arg-33, or Thr-34 decreases the ability of the nicotinamide ring to rotate. This would result in a stabilization of the nicotinamide ring in the optimum position for hydride transfer, increasing the partitioning of the 3-keto-6PG toward 6PG compared with ribulose 5-phosphate. This aspect will require further experimentation.
Results obtained for the effects of the mutations on 6PG binding are not surprising; the three residues mutated provide most of the binding energy for NADP. However, these same changes at the 2Ј-phosphate site, which is remote from the active site (ϳ15 Å), change the relative rates of hydride transfer and decarboxylation. The effect on partitioning of the intermediate provides additional, albeit indirect, evidence in support of the rotation of nicotinamide ring to facilitate decarboxylation. Data suggest that removal of any of the three residues may result in a positional change of the bound NADP, which may reorientate the nicotinamide ring of NADP to a position that disfavors ring rotation, but favors hydride transfer. This is not to say that the equilibrium constant for hydride transfer changes; rather, a change occurs in the amount of the enzyme form that undergoes the hydride transfer step.
In conclusion, the 2Ј-phosphate of NADP is critical for making critical interactions between the protein and the cofactor, helping position the cofactor for hydride transfer and participating in the rotational isomerization of the cofactor after reduction, probably by holding the cofactor in position so that the nicotinamide ring can flip into the right location. Destroying the interactions between the 2Ј-phosphate and the protein results in a slower or less efficient rotation. Attempts are now being made to obtain structures of one or more of the mutant enzymes in complex with NADP.