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Active Human Cytomegalovirus Protease Is a Dimer (∗)

      The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 μM in 10% glycerol and 0.55 μM in 20% glycerol at 30°C and pH 7.5.

      INTRODUCTION

      Viruses of the herpes family, including the human cytomegalovirus (hCMV) (
      The abbreviations used are: hCMV
      human cytomegalovirus
      Abu
      L-α-aminobutyric acid
      Dabcyl
      4-(4′-dimethylaminophenazo)benzoic acid
      Edans
      5-[(2′-aminoethyl)-amino]-naphthalene-1-sulfonic acid
      DTT
      dithiothreitol
      PAGE
      polyacrylamide gel electrophoresis
      BSA
      bovine serum albumin
      MES
      4-morpholinoethanesulfonic acid
      TAPSO
      3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid.
      ) and herpes simplex virus, encode a protease essential for viral capsid formation and viral replication (
      • Gao M.
      • Matusick-Kumar L.
      • Hurlburt W.
      • DiTusa S.F.
      • Newcomb W.W.
      • Brown J.C.
      • McCann III, P.J.
      • Deckman I.
      • Colonno R.J.
      ,
      • Preston V.G.
      • Coates J.A.V.
      • Rixon F.J.
      ,
      • Matusick-Kumar L.
      • McCann III, P.J.
      • Robertson B.J.
      • Newcomb W.W.
      • Brown J.C.
      • Gao M.
      ). The herpesvirus proteases are synthesized as precursor proteins that undergo autoproteolytic processing during viral assembly. One of the natural substrates is the viral assembly protein involved in the construction of intermediate viral capsids within the infected cell nucleus. The other natural substrate is the protease precursor protein. The protease catalytic domain is localized in the N terminus of the precursor, which in the case of hCMV encompasses the N-terminal 256 amino acids of the 708-amino acid precursor protein(
      • Welch A.R.
      • Woods A.S.
      • McNally L.M.
      • Cotter R.J.
      • Gibson W.
      ,
      • Baum E.Z.
      • Bebernitz G.A.
      • Hulmes J.D.
      • Muzithras V.P.
      • Jones T.R.
      • Gluzman Y.
      ).
      It has been suggested that the hCMV protease is a serine protease based on its chemical reactivity toward classical serine protease inhibitors (
      • Stevens J.T.
      • Mapelli C.
      • Tsao J.
      • Hail M.
      • O'Boyle II, D.
      • Weinheimer S.P.
      • DiIanni C.L
      ), and recent site-directed mutagenesis data (
      • Cox A.G.
      • Wakulchik M.
      • Sassmannshausen L.M.
      • Gibson W.
      • Villarreal W.C.
      ) have implicated the catalytic triad of hCMV protease to be Ser-132, His-63, and Glu-122. However, the catalytic efficiency of the hCMV protease is orders of magnitude less than that expected of a classical serine protease, and no amino acid sequence homology has been found between this enzyme and the well characterized serine proteases(
      • Cox A.G.
      • Wakulchik M.
      • Sassmannshausen L.M.
      • Gibson W.
      • Villarreal W.C.
      ,
      • Burck P.J.
      • Berg D.H.
      • Luk T.P.
      • Sassmannshausen L.M.
      • Wakulchik M.
      • Smith D.P.
      • Hsiung H.M.
      • Becker G.W.
      • Gibson W.
      • Villarreal E.C.
      ).
      This report identifies the dimerization of mature hCMV protease as a critical property governing its catalytic activity. Our data are consistent with a dimer dissociation constant (Kd) in the low micromolar range with the dimeric protease being the active species. Given the proposed identity of the catalytic triad and the classification of this enzyme as a serine protease, our finding marks activation by dimerization as a unique feature of this new member of the serine protease group.

      MATERIALS AND METHODS

       Enzyme Expression and Purification

      Both the wild-type and a mutant form (V141G and V207G mutations) of the hCMV protease were expressed in Escherichia coli as described previously(
      • Sardana V.V.
      • Wolfgang J.A.
      • Veloski C.A.
      • Long W.J.
      • LeGrow K.
      • Wolanski B.
      • Emini E.A.
      • LaFemina R.L.
      ). The mutations render the hCMV protease resistant to autoproteolysis. (
      V. Sardana, personal communication.
      ) All purification steps were performed at 0-4°C. For the mutant, lysis of cells was performed with a microfluidizer (Microfluidics Corp., Newton, MA) in 50 mM Tris-HCl, pH 8.0 buffer containing 10% glycerol (v/v throughout), 25 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. The lysate was centrifuged, and the pellet was washed with lysis buffer plus 0.1% Nonidet P-40 and recentrifuged. Inclusion bodies were dissolved with 7 M urea, 50 mM Tris-HCl, 5 mM DTT, pH 8.0, followed by centrifugation and chromatography on a MonoQ column (Pharmacia Biotech Inc.). Elution was performed using a sodium chloride gradient in the urea-containing buffer. Protein folding was accomplished by dilution of protease-containing fractions to 0.2 mg/ml into 25 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, 5 mM DTT, and 1 M guanidine HCl, followed by dialysis in the same buffer without guanidine HCl for 24 h. The resulting protein solution was chromatographed on a MonoQ column and eluted with a sodium chloride gradient in 25 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, and 1 mM DTT to yield the purified enzyme. The wild-type protease was purified similarly. To avoid self-proteolysis, the protein obtained from the first MonoQ step was folded and dialyzed as above, but in the absence of glycerol. The sample was then acidified to pH 5.5 with MES, applied to a Merck Fractogel SO3 column in 50 mM MES, 1 mM EDTA, 1 mM DTT, pH 5.5, and eluted with a sodium chloride gradient. The wild-type enzyme was stable at pH 5.5 and returned to full activity following dilution to pH 7.5. Enzyme preparations were greater than 95% pure by SDS-PAGE and gave the expected amino acid analysis. The N-terminal 5 residues of both enzymes were MTMDE, showing retention but deformylation of the initial N-formyl methionine. Electrospray mass spectrometry indicated a single species within 10 atomic mass units of the expected mass. The concentrations of stock enzyme solutions were determined by quantitative amino acid analysis. The data presented here were collected with use of the stable V141G/V207G mutant enzyme. The wild-type enzyme was employed to confirm all kinetic phenomena as characteristic of the hCMV protease.

       Kinetic Assays and Equilibrium Constants

      Peptide substrates used were either the fluorophore-labeled (Dabcyl)-RGVVNASSRLA-(Edans) (Bachem Biosciences, Philadelphia, PA; (
      • Holskin B.P.
      • Bukhtiyarova M.
      • Dunn B.M.
      • Baur P.
      • de Chastonay J.
      • Pennington M.W.
      )) or Ac-RWGVVNAS.Abu.RLATR-amide (Midwest Biotech, Indianapolis, IN). Products were quantified on high pressure liquid chromatography with fluorescence monitoring of the SSRLA-(Edans) product (350 nm excitation, 500 nm emission) or the Ac-RWGVVNA product (280 nm excitation, 350 nm emission). Reactions were performed for 1 min in a pH 7.5 buffer containing 52 mM MES, 52 mM TAPSO and 100 mM diethanolamine, 1 mM EDTA, 1 mM DTT, 0.05% BSA, and various concentrations of glycerol, and were quenched with the addition of urea to a final concentration of 3 M. For all of the kinetic assays conducted, hydrolysis consumed less than 15% of the substrate, and activities shown in the figures are averages of two determinations. With the exception of the Km determination, substrate concentrations during assay were 92 μM (Dabcyl)-RGVVNASSRLA-(Edans) or 87 μM Ac-RWGVVNAS.Abu.RLATR-amide. The apparent dissociation constant (Kd) for monomer-dimer equilibrium, wherein only the dimer form of the enzyme contributes to the observed velocity, was calculated with the equation
      vobs=vd. [E]t[M][E]t,
      (Eq. 1)


      where vd is the velocity for dimers, [E]t is the total concentration of enzyme (in monomer equivalents), and [M] is the concentration of monomers. The value of [M] is given by
      [M]=12[Kd2+(Kd24+2Kd[E]t)0.5]
      (Eq. 2)


      and is derived from the equilibrium condition.
      Kd=2[M]2([E]t[M]).
      (Eq. 3)


       Sedimentation Velocity

      hCMV protease was equilibrated in 100 mM sodium HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT, either with or without 20% glycerol, using Bio-Rad Biospin 6 spin columns. Samples (425 μl) were sedimented at 20°C, 45,000 rpm in a Beckman XLA analytical ultracentrifuge. Scans were recorded at 280 nm using 0.003-cm point spacing every 8 min for total run time of 4 h. The data were fit to an approximate solution of the Lamm equation using the program SVEDBERG(
      • Philo J.S.
      ). The solvent densities were measured to be 1.0063 in the absence and 1.0619 g/ml in the presence of 20% glycerol using an Anton Paar DMA48 density meter. The partial specific volume of hCMV protease, v, was calculated to be 0.727 at 20°C using the method of Cohn and Edsall (
      • Cohn E.J.
      • Edsall J.T.
      ) . Glycerol induces preferential hydration of proteins and causes v to increase. Using the data of Gekko and Timasheff, a correction factor for was found to be Δv/Δ (% volume glycerol) = 3.33 ± 0.38 × 10-4(average of 4 proteins)(
      • Gekko K.
      • Timasheff S.N.
      ).

       Size Exclusion Chromatography

      Either a Pharmacia Superdex 75 column (300 × 10 mm) or two Bio-Rad BioSelect 125 columns (300 × 7.4 mm) connected in series were used immersed in a controlled-temperature water bath. Chromatography with the Superdex column was at 20°C in buffer (0.1 M sodium HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT) containing either 0% or 20% glycerol, and sample volumes injected were 200 μl. Chromatography with the BioSelect columns was at 10°C except where indicated, and in pH 7.5 buffer containing 52 mM MES, 52 mM TAPSO, 100 mM diethanolamine, 1 mM EDTA, and 20% glycerol (assay buffer without BSA). For Kd determinations, samples were maintained at 30°C in a controlled temperature sample compartment prior to loading onto columns equilibrated in 20% glycerol-containing buffer at 10°C. Detection of eluting hCMV protease was by simultaneous monitoring of absorbance at 280 nm and fluorescence (excitation 280 nm, emission 350 nm). Comparison of integrated peak areas using the two methods showed a consistent 1.26-fold higher fluorescence response for monomers than for dimers, so that calculations of total protein in the monomer fluorescence peaks were adjusted accordingly. The absorbance extinction coefficients at 280 nm for monomers and dimers were assumed to be equal. The peak shapes and intensities obtained were unchanged for samples preincubated at 30°C from 1.5 to 8 h. Column buffers were degassed with and maintained under helium. Proteins used for standardization were BSA (64 kDa), ovalbumin (43 kDa), chymotrypsinogen (23.2 kDa), myoglobin (16.9 kDa), and bovine pancreatic RNase (12.6 kDa). Complete sets of standards were run at the beginning, the end, and in the middle of each solvent study, with little variation in elution volumes.

      RESULTS

       Dependence of Protease Specific Activity on Enzyme and Glycerol Concentrations

      Dilution of a concentrated solution of hCMV protease produces a time-dependent change in activity to a new, lower level. The rate and extent of the change in protease activity is a function of temperature. At 37°C, the decrease in protease activity occurs within minutes, while at 0°C, no significant change is observable for a period of hours. The change in hCMV protease activity at four temperatures upon dilution as a function of time is shown in Fig. 1. At temperatures above 20°C, the change in activity upon enzyme dilution is rapid enough (t½ ≤ 30 min) that an accurate assessment of activity requires a short assay (~1 min).
      Figure thumbnail gr1
      Figure 1:Activity of the hCMV protease as a function of time after dilution at 0, 25, 30, and 37°C. A 2.5 μM hCMV protease sample (monomer equivalents) at 0°C was diluted 50-fold to 50 nM into a buffer (52 mM MES, 52 mM TAPSO, 100 mM diethanolamine, 1 mM EDTA, 1 mM DTT, 20% glycerol, 0.05% BSA, pH 7.5) at the temperature indicated, and at the times shown an aliquot was withdrawn and assayed with the substrate Ac-GVVNAS.Abu.RLATR-amide at the same temperature. Assays were performed for a period of 1 min as described under “Materials and Methods.”
      The specific activity of the hCMV protease increases at higher enzyme and glycerol concentrations. These effects at 30°C are shown in Fig. 2. The specific activities of the protease measured after enzyme dilution and incubations of 1.5 and 3.5 h prior to reaction are shown in Fig. 2A. The data reveal that the hCMV protease specific activity tends toward zero as its concentration is lowered. The negligible difference between the determinations at 1.5 and 3.5 h suggests that the active form of the enzyme has reached equilibrium within 1.5 h, for both the 10% and 20% glycerol samples. The activity of the protease incubated in 10% glycerol (v/v) is lower than that incubated in 20% glycerol (v/v). It can be shifted back to the higher activity seen for 20% glycerol by addition of an equal volume of buffer containing 30% glycerol to produce a solution containing 20% glycerol, followed by further incubation. Thus, the dependence of hCMV protease activity on enzyme and glycerol concentrations is reversible. The reversal effect is shown in Fig. 2B.
      Figure thumbnail gr2
      Figure 2:Specific activity of the hCMV protease as a function of protein and glycerol concentrations. A, enzyme solutions at the concentrations indicated (monomer equivalents) were incubated at 30°C in assay buffer (50 μl) before initiating a 1-min assay with the addition of 30 μl of the peptide substrate (Dabcyl)-RGVVNASSRLA-(Edans) in the same buffer. Results for incubations of 1.5 h (•) and 3.5 h (■) in 10% glycerol (v/v) and 1.5 h (◆) and 3.5 h (▵) in 20% glycerol (v/v) are shown. B, samples of the enzyme dilutions incubated at 30°C for 1.5 h in 10% glycerol were mixed with an equal volume of buffer containing 30% glycerol (v/v) to give a final glycerol concentration of 20% and incubated for an additional 2 h. Assays of these samples were conducted alongside those incubated continuously in 20% glycerol. The data for the 10%-shifted-to-20% glycerol samples (○) are plotted with those of the 3.5-h incubations shown in panel A in 10% (□) and 20% (▵) glycerol. The enzyme concentrations indicated on the abscissa are those obtained before assay initiation. The solid lines in both panels correspond to fits of a monomer-dimer equilibrium relationship (see “Materials and Methods” and “Discussion”) wherein the monomer is inactive. The Kd values of 6.2 μM (1.5 h) and 6.9 μM (3.5 h) were obtained in 10% glycerol and Kd values of 0.58 μM (1.5 h) and 0.51 μM (3.5 h) were obtained in 20% glycerol. The 10%-shifted-to-20% fit produced a Kd of 0.84 μM. The average vd, or velocities for fully dimerized enzyme under these conditions (see “Materials and Methods”), are 430 nmol min-1 mg-1 for 10% glycerol and 400 nmol min-1 mg-1 for 20% glycerol.
      The kinetic parameters Vmax and Km for the substrate (Dabcyl)-RGVVNASSRLA-(Edans) were determined with different hCMV protease concentrations preincubated in 20% glycerol using assay conditions as in Fig. 2. The Vmax and Km values obtained with 0.5 μM enzyme are 360 nmol min-1 mg-1 and 92 μM, respectively, while for 2.0 μM enzyme they are 600 nmol min-1 mg-1 and 97 μM. Thus it is the apparent turnover rate of the enzyme that varies with enzyme concentration.

       Analytical Centrifugation

      The quaternary state of the hCMV protease was characterized by sedimentation velocity measurements. Table 1 shows the results of sedimentation analyses of samples at a concentration of 20 μM in the presence or absence of 20% glycerol (v/v). An approximate solution of the Lamm equation can be fit directly to the data to obtain the sedimentation coefficient, s20,w, the diffusion coefficient, D20w, and by use of the Svedberg relation, the molecular weight(
      • Philo J.S.
      ). For both samples good fits are found for a single sedimenting species. In the absence of glycerol the molecular weight obtained is 29.7 × 103, which is close to the molecular weight of a monomeric hCMV protease. In 20% glycerol, 48.5 × 103 is obtained, indicating that the enzyme is in a predominantly dimeric form. (
      Similar results (J. Cole, unpublished data) were obtained from equilibrium sedimentation measurements. At protein concentrations below 1 μM, we find that in the absence of glycerol the molecular weight of the hCMV protease is within 5% of the monomer value. In the presence of 20% glycerol, the apparent weight-average molecular weights increase at intermediate protein concentrations and then decrease at higher concentrations; this behavior is typical of a dimerizing protein which exhibits thermodynamic nonideality(
      • Johnson M.L.
      • Correia J.J.
      • Yphantis D.A.
      • Halvorson H.R.
      ). The sedimentation equilibrium data obtained in the presence of 20% glycerol at a protease concentration of 50 μM fit to a nonideal dimer model giving an apparent molecular weight of 52,100. A thorough sedimentation equilibrium study will be presented separately.
      ) The increase in s20w and the decrease in D20w shown in Table 1 are both consistent with the existence of a stable dimeric protease in 20% glycerol.
      Tabled 1
      Table thumbnail fxt1

       Size Exclusion Chromatography

      The hCMV protease preincubated in the absence of glycerol and applied to a size exclusion column elutes with an apparent molecular weight of 37 × 103 or 56 × 103, depending the loading concentration of the protease. With 20% glycerol present during preincubation, the protease elutes as a single 56 × 103 species except with relatively low protease loading concentrations (<10 μM), where two fractions emerge with apparent weights of 55 × 103 and 33 × 103, as summarized in Table 2. Since the molecular weight of the hCMV protease calculated from amino acid sequence is 28 × 103, these results suggest that the protease exists in a monomer-dimer equilibrium. Eluted enzyme samples corresponding to a dimeric protease show no evidence of covalent (disulfide) linkages as demonstrated by SDS-PAGE under non-reducing conditions. The elution profiles obtained from samples equilibrated in 20% glycerol at 30°C, analyzed on the BioSelect columns run at 10°C in 20% glycerol, are shown in Fig. 3A.
      Tabled 1
      Table thumbnail fxt2
      Figure thumbnail gr3
      Figure 3:Size exclusion chromatography of the hCMV protease. Samples of hCMV protease in 20% glycerol were maintained at 30°C for at least 90 min prior to chromatography on two Bio-Rad BioSelect columns (in tandem) at 10°C, as described under “Materials and Methods.” A, elution profiles for samples at concentrations, prior to injection, of 171 nM (a), 355 nM (b), 891 nM (c), 1975 nM (d), 2977 nM (e), and 4501 nM (f). Shown here are the protease fluorescence emission data at 350 nm. Injection volumes were adjusted to give the same total protein injected (30 pmol, monomer equivalents). B, fraction of total protein appearing in the dimer peak (26.2 min) as a function of protein concentration. The solid line corresponds to a fit of the monomer-dimer equilibrium function (see “Materials and Methods”) to the data, yielding a Kd of 0.54 μM, with a maximum dimer fraction of 0.92. In separate experiments using a loading concentration of 100 μM protease, the maximum fraction of dimeric protease is ≥0.95, suggesting that all the hCMV protease in our purified sample is fully capable of dimerization.
      Assignment of the early and late elution peaks in Fig. 3A as dimer and monomer, respectively, allows the estimation of a Kd for dimerization. Using the ratios of the areas under-the-peak of the early and late peaks, a Kd value of 0.54 μM is found for 20% glycerol as shown in Fig. 3B. The maximum fraction of dimeric protease extrapolated from Fig. 3B is 0.92. In separate experiments using protease at a loading concentration of 100 μM, the maximum fraction of dimeric protease is ≥0.95. When activity assays are conducted at 0°C, no detectable activity is found in the eluted peak corresponding to the monomer while hydrolytic activity (>50-fold of detectable level) is found for the dimer peak. The same analysis applied to enzyme pre-equilibrated in 10% glycerol produces a Kd of 5.5 μM. The activity data sets shown in Fig. 2 can now be justifiably treated with a model wherein an inactive monomeric hCMV protease exists in equilibrium with an active dimeric hCMV protease. Fits of this model (see “Materials and Methods”) to the kinetic data (Fig. 2) give average dissociation constants (Kd) for hCMV protease of 6.6 μM in 10% glycerol and 0.55 μM in 20% glycerol.
      Changes in sample loading volume, column temperature, and chromatography time have been made to affirm that equilibrium exchange between protease monomers and dimers is negligible during size exclusion chromatography at 10°C in 20% glycerol. No significant variation in dimer-monomer peak ratios occurs when injection volumes of 5, 10, 20, or 30 μl (15-90 pmol) of protease sample are made. Column temperatures of 5, 10, and 18°C produce essentially identical results as well. Some coalescence of elution peaks toward the monomeric form is observed at 25°C, and complete peak merging occurs at 30°C to yield mostly monomer. With the chromatography temperature at 10°C, as in the analyses presented here, the dimer-monomer ratio has also been compared for the use of one versus two sizing columns. While the resolution with two columns in tandem (shown in Fig. 2A) is slightly better than with one column alone, the dimer-monomer ratio observed is identical, despite the fact that chromatography runs are completed in one-half the time with the single column. We conclude that despite an approximate 100-fold dilution of sample during the chromatography at 10°C, the aggregation state of the sample upon injection is well approximated by the elution patterns observed, due to the slow monomer-dimer equilibration at low temperatures.

      DISCUSSION

      This report describes our findings that the hCMV protease activity is dependent upon protein dimerization. That the activity of the protease is a function of time after dilution (Fig. 1) and of enzyme concentration (Fig. 2) suggests the existence of a protomer-oligomer equilibrium with the oligomeric enzyme being the active species. Physical data from sedimentation velocity and size exclusion chromatography experiments are evidence that the multimeric form of the protease is a dimer. Sedimentation velocity data (Table 1) obtained in the absence of glycerol gives 29.7 × 103 as the molecular weight of the hCMV protease, as expected for a monomeric enzyme species. In the presence of 20% glycerol, molecular weights obtained from sedimentation velocity (Table 1) and sedimentation equilibrium3 runs are consistent with a dimeric protease. The molecular weights estimated from size exclusion chromatography for the two enzyme species (Table 2) are relative values that depend upon the calibrating proteins used, but they are in agreement with sedimentation results. Most importantly, the conversion of the protease from dimers to monomers is quantitatively reversible (Fig. 2B). The isolated monomer is found to be inactive, when tested at 0°C to prevent re-equilibration, while its dimeric counterpart is active (Fig. 3). These results, when considered together, are consistent with a simple monomer-dimer equilibrium model in which only the dimeric protease is the active form ().
       KdM+MDD+SKSD. SKcatD+P1+P2D+SKcat/KMD+P1+P2


      Although only the dimer form of the protease is depicted to bind substrates in, (
      As depicted, substrates are expected to enhance dimerization of the protease. This phenomenon has been observed with specific peptide substrates of the hCMV protease but not with peptides that are not substrates of the protease (P. L. Darke, unpublished observations).
      ) it is not possible for us to state unequivocally that the monomeric form of the enzyme does not bind substrate or does not possess a very low catalytic activity. Also unclear at present is the stoichiometry of substrate binding to the enzyme. Our efforts continue in clarifying these issues.
      further defines the kinetic parameters of the hCMV protease based on the results reported here. Determinations of the Kd can be complicated during substrate turnover by the interchange of enzyme forms on the minute time scale (Fig. 1), so a short reaction time is required for accurate estimates. Our kinetic results (Fig. 2) indicate that the affinity of the monomeric protease for itself at 30°C is weak with a Kd in the low micromolar range: 6.6 and 0.54 μM in the presence of 10% and 20% glycerol, respectively. Essentially the same Kd values, 5.5 and 0.55 μM, respectively, are obtained from size exclusion measurements (Fig. 3B). Kinetic estimates of Kd with the alternate substrate Ac-RWGVVNAS.Abu.RLATR-amide are in complete agreement with these values (data not shown).
      Given and the rate of activity relaxation at 30°C shown in Fig. 1, the 1-min assay measurements should approximate the dimer catalytic activity prior to subunit dissociation by dilution and prior to substrate perturbation of the monomer-dimer equilibrium. The Vmax measurements then allow calculation of a kcat for fully dimeric enzyme using the assumption of a single active site per subunit. The Vmax values in 20% glycerol of 360 and 600 nmol min-1 mg-1 for 0.5 and 2.0 μM total enzyme, respectively, combined with the corresponding fractions of enzyme present as dimer calculated from the Kd, 0.48 and 0.69, give similar kcat values of 21.2 and 24.2 min-1. The velocity of dimeric enzyme, vd, expected to be found in the enzyme titration of Fig. 2 can be calculated from the average of these kcat values, 22.7 min-1, taking consideration of substrate concentration used (92 μM) relative to the average Km under these conditions (94 μM) to yield vd equal to 401 nmol min-1 mg-1. In fact, vd from the curve fit of data in Fig. 2 yields 400 nmol min-1 mg-1 in 20% glycerol. The true kcat/Km value for this dimeric enzyme with (Dabcyl)-RGVVNASSRLA-(Edans) at 30°C in 20% glycerol is therefore 4000 M-1 s-1, several times higher than previously reported for this and related substrates(
      • Burck P.J.
      • Berg D.H.
      • Luk T.P.
      • Sassmannshausen L.M.
      • Wakulchik M.
      • Smith D.P.
      • Hsiung H.M.
      • Becker G.W.
      • Gibson W.
      • Villarreal E.C.
      ,
      • Sardana V.V.
      • Wolfgang J.A.
      • Veloski C.A.
      • Long W.J.
      • LeGrow K.
      • Wolanski B.
      • Emini E.A.
      • LaFemina R.L.
      ,
      • Holskin B.P.
      • Bukhtiyarova M.
      • Dunn B.M.
      • Baur P.
      • de Chastonay J.
      • Pennington M.W.
      ).
      In summary, the data presented here reveal that the hCMV protease exists as a dimeric species under conditions that enhance the specific activity of the enzyme, such as high enzyme concentration or the presence of glycerol. The activating effect of glycerol has been noted previously(
      • Burck P.J.
      • Berg D.H.
      • Luk T.P.
      • Sassmannshausen L.M.
      • Wakulchik M.
      • Smith D.P.
      • Hsiung H.M.
      • Becker G.W.
      • Gibson W.
      • Villarreal E.C.
      ,
      • Pinko C.
      • Margosiak S.A.
      • Vanderpool D.
      • Gutowski J.C.
      • Condon B.
      • Kan C.-C.
      ), but not the underlying mechanism of activation. Activity of the protease observed in assays containing no glycerol is likely due to a small concentration of dimers in equilibrium with monomers. Although unusual for a serine protease, this mode of activation may provide an appropriate temporal trigger for proteolytic activity during the assembly of hCMV capsids, a process wherein the protein components become highly concentrated.

      Acknowledgments

      We thank Dr. Robert LaFemina for the hCMV protease expression plasmids and Dr. John Philo for the SVEDBERG program.

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