(Received for publication, January 17, 1996; and in revised form, February 1, 1996)
From the
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.
Viruses of the herpes family, including the human
cytomegalovirus (hCMV) ()and herpes simplex virus, encode a
protease essential for viral capsid formation and viral replication (1, 2, 3) . 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(4, 5) .
It has been suggested that the hCMV protease is a serine protease based on its chemical reactivity toward classical serine protease inhibitors (6) , and recent site-directed mutagenesis data (7) 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(7, 8) .
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 (K) 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.
where v is the velocity for dimers,
[E]
is the total concentration of enzyme
(in monomer equivalents), and [M] is the
concentration of monomers. The value of [M] is given
by
and is derived from the equilibrium condition.
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 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 K
values of 6.2 µM (1.5 h) and 6.9 µM (3.5 h) were obtained in 10%
glycerol and K
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 K
of 0.84 µM. The average v
,
or velocities for fully dimerized enzyme under these conditions (see
``Materials and Methods''), are 430 nmol min
mg
for 10% glycerol and 400 nmol
min
mg
for 20%
glycerol.
The kinetic
parameters V and K
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 V
and K
values obtained with 0.5 µM enzyme
are 360 nmol min
mg
and 92
µM, respectively, while for 2.0 µM enzyme
they are 600 nmol min
mg
and 97
µM. Thus it is the apparent turnover rate of the
enzyme that varies with enzyme concentration.
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 K 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 K for dimerization.
Using the ratios of the areas under-the-peak of the early and late
peaks, a K
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 K
of
5.5 µM. The activity data sets shown in Fig. 2can
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 (K
) 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.
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
10
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 equilibrium
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 ().
Although only the dimer form of the protease is depicted to bind
substrates in , ()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 K 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 K
in the low micromolar range: 6.6 and 0.54
µM in the presence of 10% and 20% glycerol, respectively.
Essentially the same K
values, 5.5 and 0.55
µM, respectively, are obtained from size exclusion
measurements (Fig. 3B). Kinetic estimates of K
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 V measurements then allow calculation of a k
for fully dimeric enzyme using the assumption of a single active
site per subunit. The V
values in 20% glycerol
of 360 and 600 nmol min
mg
for
0.5 and 2.0 µM total enzyme, respectively, combined with
the corresponding fractions of enzyme present as dimer calculated from
the K
, 0.48 and 0.69, give similar k
values of 21.2 and 24.2
min
. The velocity of dimeric enzyme, v
, expected to be found in the enzyme titration of Fig. 2can be calculated from the average of these k
values, 22.7 min
, taking
consideration of substrate concentration used (92 µM)
relative to the average K
under these conditions
(94 µM) to yield v
equal to 401 nmol
min
mg
. In fact, v
from the curve fit of data in Fig. 2yields 400 nmol
min
mg
in 20% glycerol. The true k
/K
value for this dimeric
enzyme with (Dabcyl)-RGVVNASSRLA-(Edans) at 30 °C in 20% glycerol
is therefore 4000 M
s
,
several times higher than previously reported for this and related
substrates(8, 9, 10) .
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(8, 15) , 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.