©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processive Post-translational Modification
VITAMIN K-DEPENDENT CARBOXYLATION OF A PEPTIDE SUBSTRATE (*)

(Received for publication, June 13, 1995; and in revised form, September 27, 1995)

Daniel P. Morris (1) Robert D. Stevens (2) David J. Wright (1)(§) Darrel W. Stafford (1)(¶)

From the  (1)Department of Biology and the Center for Thrombosis and Hemostasis, University of North Carolina, Chapel Hill, North Carolina 27599-3280 and the (2)Division of Biochemical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive post-translational modification (i.e. multiple carboxylations occur during a single association between enzyme and substrate). Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids -18 to 41 of the human blood clotting enzyme factor IX. Mass spectrometry was used to determine the number of -carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction. Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times. This observation can only be explained by two types of mechanisms. In a processive mechanism, multiple carboxylations could occur during a single substrate binding event. Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S. Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translational modification.


INTRODUCTION

The vitamin K-dependent carboxylase converts glutamate to carboxyglutamate (Gla) by the addition of CO(2) at the -position(1, 2, 3, 4, 5) . In addition to glutamate and CO(2), the reaction requires the cosubstrates O(2) and reduced vitamin K. The native substrates for the carboxylase include several proteins central to blood clotting as well as bone Gla protein, matrix Gla protein, and others. Each of these proteins contain a ``Gla domain'' in which glutamate is converted to Gla. This Gla domain binds Ca and is essential for activity in all of the identified Gla-containing proteins.

Efficient carboxylation of the native substrates requires that they contain a conserved region which binds to the carboxylase with a submicromolar affinity constant(6, 7) . In the blood-clotting factors, this region is contained in an amino-terminal propeptide which is proteolytically removed following carboxylation. Substrates containing this propeptide have K values 3 to 4 orders of magnitude lower than small peptide substrates that do not contain the propeptide(8, 9, 10) . This suggests that the propeptide acts primarily as a ``docking'' site which localizes the glutamate residues near the carboxylase active site.

Using the vitamin K-dependent carboxylase activity present in solubilized bovine microsomes, we previously reported the introduction of multiple Gla residues into peptide substrates derived from the propeptide and Gla domain of human Factor IX(9) . In this study, we use one of those substrates, FIXQ/S (Fig. 1), to investigate the mechanism by which multiple glutamates per substrate are converted to Gla residues during vitamin K-dependent carboxylation. As a consequence of our recent purification of the vitamin K-dependent bovine carboxylase(11) , we can perform in vitro carboxylations from which the carboxylated products can easily be recovered. After suitable purification, the number of Gla residues introduced into the peptide products can be determined by mass spectrometry on the products as they elute from a reverse phase HPLC (^1)column. We also found that elution of the various carboxylation states from an anion exchange HPLC column is closely correlated with the extent of carboxylation. This behavior allowed us to perform a series of kinetic experiments to demonstrate that the vitamin K-dependent carboxylase is truly processive.


Figure 1: Sequence of the human FIX-based substrate FIXQ/S. The propeptide and Gla domains are separated by the tilde () symbol. The 12 glutamates that are converted to Gla in the native Factor IX are in bold type. Although these changes are unlikely to affect carboxylation, residues that have been mutated from the native Factor IX are underlined.



Examples of processive enzymes are very common in the replication and modification of RNA and DNA(12) . However, examples of processive post-translational modification for which there is good supporting evidence appear to be rare. Nevertheless, processive modes of post-translational modification should offer significant advantages in a wide variety of biochemical processes where multiple identical modifications are introduced into a substrate. The few apparent cases of such modifications in the scientific literature reflect this diversity and suggest that processive post-translational modification may be very common. Unlike processivity involving nucleic acids, processive post-translational modification of proteins may often occur in patterns other than a linear sequence along the polymer chain. The vitamin K-dependent -glutamyl carboxylase appears to be an excellent model for studying such processes.


MATERIALS AND METHODS

Chemicals

Vitamin K(1) (phylloquinone) at 10 mg/ml was obtained from Abbott Laboratories in the form of a colloidal suspension (Vitamin K(1) Injection) which included 7% polyoxylated fatty acid, 3.75% hydrous dextrose, and 0.9% benzyl alcohol. Phosphatidylcholine, type X-E, from dried egg yolk, and CHAPS were from Sigma. NaH^14CO(3) with a specific activity of 56.4 mCi/mmol was from ICN Pharmaceuticals Inc.

Preparation of Reaction Materials

Carboxylase was affinity-purified as described previously except that either FIXQ/S or proFIX19 was used as the propeptide stabilizer of carboxylase(11) . The fractions of carboxylase used in this study were stored at -70 °C and contained 1.2-2 µM FIXQ/S or proFIX19, 0.6-1% CHAPS, 0.1% phosphatidylcholine, 25 mM MOPS (pH 7.4), 1 times protease inhibitor mixture, 500 mM NaCl, and 20% (v/v) glycerol. The carboxylase was eluted with FIXQ/S unless the presence of propeptide is indicated. Carboxylase was quantitated by densitometric scanning of Coomassie-stained gels using IgG as a standard. Molar concentrations of carboxylase were calculated using a molecular mass of 88 kDa(13) .

The peptide substrate FIXQ/S was expressed in Escherichia coli as a fusion protein and cleaved with cyanogen bromide as described previously(9) . The peptide products dissolved in guanidine HCl were dialyzed against 25 mM MOPS (pH 8.0) with 50 mM NaCl. The soluble FIXQ/S product was collected as described previously(9) . Formylation of cysteine was reversed by incubating FIXQ/S with 20 mM DTT for 4 h at 25 °C(14) . The peptide-containing solution was then chilled to 4 °C and loaded onto a DEAE-Sepharose column (20 ml of matrix per 50 mg of protein) equilibrated with 25 mM MOPS (pH 8.0) and 50 mM NaCl. The column was washed with 2 volumes of equilibration buffer, and the protein eluted in 25 mM MOPS (pH 8.0) with a NaCl gradient from 50 to 500 mM NaCl.

Vitamin K(1) (Abbott Laboratories) at 10 mg/ml was reduced by addition to 3 volumes of buffer containing 200 mM DTT, 500 mM NaCl, and 20 mM Tris (pH 8.5). Full tubes were incubated in darkness at 4 °C for at least 36 h to ensure complete reduction.

Preparative Carboxylase Reactions

After all additions, reactions to generate sufficient carboxylated FIXQ/S for mass spectrometry included: 5-20 nM carboxylase, 1.3-1.4 µM FIXQ/S, 0.38 mM NaH^14CO(3), 25 mM MOPS (pH 7.4), 100 mM NaCl, 0.12% phosphatidylcholine, 0.26-0.3% CHAPS, 4% (v/v) glycerol, 0.4 mM EDTA, and 6 mM DTT. Typically, 75 µM vitamin KH(2) was added initially, and an additional 37 µM vitamin KH(2) was added at 12 h. These later additions increased the DTT concentration slightly above 6 mM. Reactions up to 500 ml were done.

To perform a reaction, most of the solution components were combined and placed on ice. After the solution had chilled, purified carboxylase which represented 20% of the final reaction volume was thawed and added to the ice-cold solution. The NaH^14CO(3) was then added, and the solution was placed in a 17 °C water bath for a 1-h preincubation. The reaction was initiated by addition of vitamin KH(2) and allowed to proceed for as long as 44 h at 17 °C.

The progress of the reaction was followed by periodically removing small samples and assaying them for incorporated NaH^14CO(3) as in previous FLEEL assays(15) . At the desired time, large reactions were terminated in a fume hood by the addition of acetic acid to a final concentration of 30 mM, resulting in a pH below 5. The remaining steps were done at room temperature in a fume hood. The NaH^14CO(3) was removed by bubbling N(2) gas through the reaction solution for about 30 min. The ^14CO(2) gas was collected by running the exhaust gas through two consecutive traps containing 0.5 N NaOH. The reaction container was sealed except for tubes leading from the nitrogen and to the NaOH trap. The tube from the nitrogen went to the bottom of the reaction vessel and could be used to remove small amounts of solution. After scintillation counting revealed that more than 99.9% of the free radioactivity had been removed, reaction solutions were neutralized by addition of NaOH to a final concentration of 30 mM and diluted with 1.5 volumes of deionized water. The diluted reaction was combined with 0.04 volume of a 50% slurry of DEAE-Sepharose in 10 mM Tris, pH 7.3, and 50 mM NaCl. The matrix was recovered in minimal volume and loaded into a 2.5-cm diameter column. The column was washed with 2 volumes of column equilibration buffer and eluted with 10 mM Tris (pH 7.3) and 1 M NaCl. Fractions containing ^14C-FIXQ/S were determined by scintillation counting a portion of each sample. These fractions were then pooled and frozen. In all experiments and at all stages, peptide samples were frozen in liquid nitrogen and stored at -70 °C. All fractions were collected and stored in polypropylene Microfuge tubes.

HPLC Purification of Carboxylated FIXQ/S

After thawing, a sample was concentrated and desalted on Centricon 3 concentrators (Amicon). This desalted sample was centrifuged (13,000 times g) to remove particulates and loaded onto a 0.46 times 100 mm Rainin Hydropore AX weak anion exchange column equilibrated with 100% buffer A (20 mM Tris, pH 7.2). A sample was loaded using multiple injections over 5-10 min while the flow rate was 1 ml/min. Subsequently, the flow rate was linearly reduced over 1 min to 0.5 ml/min, and the column eluted at 0.5 ml/min as follows: linear gradient to 30% buffer B (20 mM Tris (pH 7.2) and 2 M NaCl) over 12 min, linear gradient to 100% B over 5 min, then at least 10 min at 100% B. Fractions containing radioactivity were pooled, frozen, and stored.

To remove uncarboxylated FIXQ/S, fractions containing labeled FIXQ/S were concentrated, desalted, and fractionated a second time on the Hydropore AX column as described above. Eluted fractions were then pooled into groups and frozen. During the above procedures, protein was quantitated from peak area relative to known amounts of FIXQ/S using absorbance at 220 nm.

After thawing, each group was centrifuged to remove particulates and loaded directly onto a 0.46 times 50 mm Rainin C18 column (83-203-F5) with matching guard column. Samples were loaded using multiple injections over 10 min during which buffer A (0.1% trifluoroacetic acid) was flowing at 1 ml/min. After the load period, the flow rate was linearly reduced over 1 min to 0.125 ml/min followed by 2 min at this velocity. Next, a linear elution gradient brought the mobile phase composition to 100% B (0.1% trifluoroacetic acid, 90% acetonitrile) over 10 min. The remaining 15 min of each run was 100% B. Protein-containing fractions were collected, frozen in liquid nitrogen, and dried by rotary evaporation. Protein was quantitated from peak area relative to known amounts of FIXQ/S using absorbance at either 220 or 280 nm. Each group was resuspended to 1 µg/µl in 25 mM ammonium hydroxide after which acetic acid was added to a final concentration of 15 mM. Each group was aliquoted and frozen.

On-line Mass Spectrometry

Immediately prior to use, a 10-µl aliquot from each group was thawed and warmed to room temperature. To the sample was added in order: 1 µl of 200 mM DTT, 1 µl of 200 mM ammonium hydroxide, and 1 µl of 20 mM EDTA. A portion of each sample (5 µl) was loaded onto the 2.1 times 150 mm Vydac C4 column (214TP 5215) equilibrated with 90% solution A (0.025% trifluoroacetic acid) and 10% solution B (0.25% trifluoroacetic acid, 90% acetonitrile). The protein was eluted using an ISCO microbore HPLC apparatus at 20 µl/min according to the following gradient: 10% B from 0 to 10 min, linear gradient to 50% B from 10 to 15 min, linear gradient to 90% B from 15 to 40 min, and 90% B from 40 to 45 min. The effluent was monitored at 216 nm and run directly into the mass spectrometer through PEEK tubing (inside diameter 0.0051 cm).

Mass measurements were made on a Fisions-VG Quattro BQ triple quadropole mass spectrometer equipped with a pneumatically assisted electrostatic ion source operating at atmospheric pressure. Mass spectra were acquired in the continuum mode from m/e 700-1400 with a scan time of 10 s. The mass scale was calibrated with horse heart myoglobin (16951.48 kDa) with a resolution corresponding to a peak width at half-height of 1.4 Da for m/e 893. The mass spectra were transformed to a molecular mass scale using software supplied by the manufacturer.

The average carboxylation state of carboxylated products was estimated using the assumption that the ionization efficiencies of all carboxylation states within a group were the same. From the relative amounts of each carboxylation state in the mass spectrometry profile for a group, the average carboxylation state of the carboxylated products in the group was estimated. The average carboxylation state for each group and the amount of carboxylated peptide in each group was then used to determine the overall average carboxylation state of the product peptides.

Nonpreparative Carboxylase Reactions

Changes from the preparative reaction procedure are indicated in the figure legends. For these reactions, recovery of carboxylated and uncarboxylated FIXQ/S by DEAE-Sepharose was done in 15- or 50-ml polypropylene tubes. Low speed centrifugation (2,000 times g) of these tubes allowed essentially complete recovery of the matrix.

The carboxylation state of the reaction products from these experiments was estimated from their elution position on the Hydropore AX anion exchange column. For these experiments, the preparative anion exchange HPLC method was modified to the extent that elution gradients were linear from 0-100% buffer B and occurred over 25 min at a flow rate of 1 ml/min.


RESULTS

Preparative Carboxylation Reactions

Following an initial burst of activity, carboxylation of the substrate FIXQ/S (Fig. 1) by the purified vitamin K-dependent carboxylase was linear with respect to time for more than 24 h provided the reaction was supplemented periodically with vitamin KH(2) (Fig. 2). During this linear period, the average reaction velocity was 10 s, indicating that one carboxylation occurs per enzyme about every 17 min.


Figure 2: Time course of carboxylation of FIXQ/S is linear following an initial burst of activity. The 350-ml reaction shown included 1,300 nM FIXQ/S and 20 nM carboxylase. Projecting the linear portion of the time course to the axis suggests an apparent burst size of 350 nM. Mass spectrometry data presented below indicates that the specific activity of the incorporated NaH^14CO(3) was 40.3 mCi/mmol. This value was used to calculate the incorporated ^14CO(2) concentration. After 27 h of incubation, both the carboxylated and uncarboxylated FIXQ/S from this experiment were recovered as described under ``Materials and Methods.''



In enzymatic reactions, a rapid burst of product formation followed by a slower linear rate of reaction often indicates that product release is rate-limiting for the reaction. These burst phenomena are a consequence of the accumulation of product-enzyme complex from a single substrate binding event and catalysis. In this situation, the size of a burst may indicate the concentration of active enzyme in the reaction and can be determined by linear extrapolation of the linear portion of the progress curve back to zero time. For the experiment in Fig. 2, this method suggests a burst size of 350 nM incorporated CO(2) which is well above the 20 nM carboxylase concentration estimated to be present in the reaction. However, it is reasonably close to the concentration of CO(2) one would expect to incorporate into 20 nM FIXQ/S if an average of about 7.5 CO(2) molecules are incorporated per FIXQ/S molecule (e.g. 150 nM). Data presented later suggest that this average is approximately correct. Imprecision in the quantitation of the carboxylase, which was done on SDS gels by comparison with IgG, could easily account for the remaining difference. The presence of a burst phenomenon which could be explained by multiple carboxylations per binding event suggests a processive reaction with a rate-limiting product release step.

Recovery of FIXQ/S

The FIXQ/S substrate and the carboxylated peptides were simultaneously recovered from reaction mixtures by batch chromatography on DEAE-Sepharose (see ``Materials and Methods''). Recovery of the peptides and the incorporated radioactivity were essentially quantitative when care was taken to recover all of the Sepharose matrix.

Anion Exchange HPLC Resolves Carboxylated and Uncarboxylated Peptides

Because the carboxylated and uncarboxylated peptides were recovered together, it was necessary to separate the much more abundant uncarboxylated FIXQ/S from the carboxylated products if these products were to be analyzed effectively by mass spectrometry. The carboxylated and uncarboxylated forms of FIXQ/S were found to be relatively well separated by weak anion exchange HPLC (Fig. 3). Nevertheless, enough uncarboxylated FIXQ/S trailed the uncarboxylated FIXQ/S peak that it was necessary to combine fractions containing labeled peptide from the initial anion exchange elution and chromatograph them a second time. The absorbance profile from the second of two such sequential anion exchange runs is shown in Fig. 4. For these runs, a low flow rate and steep gradient were used to elute the carboxylated products in a minimum volume.


Figure 3: Anion exchange HPLC on post-reaction FIXQ/S separates the carboxylated and uncarboxylated forms. A small portion (0.5%) of the FIXQ/S from the reaction in Fig. 2was loaded on a Rainin Hydropore HA weak anion exchange column and eluted with a gradient of the composition indicated. Buffer A contained 20 mM Tris, pH 7.2, while buffer B additionally included 2 M NaCl. For more detail, see ``Materials and Methods.''




Figure 4: Consecutive anion exchange column runs reduce the amount of uncarboxylated FIXQ/S that coelutes with carboxylated forms of FIXQ/S. FIXQ/S from the reaction in Fig. 2was desalted and loaded onto the Rainin Hydropore HA anion exchange column (data not shown). Carboxylated FIXQ/S fractions containing fractions from the first anion exchange elution (as indicated by the presence of radioactivity) were combined, desalted, and rerun as before. The FIXQ/S from the second elution (shown above) was pooled into six groups labeled G1 to G6 as indicated. Quantitation during reverse phase HPLC indicate that the groups contained the following amounts of protein: G2, 44 µg; G3, 33 µg; G4, 20 µg; G5, 40 µg; G6, 143 µg. See ``Materials and Methods'' for details.



Since increasing carboxylation of a peptide would result in a more negatively charged product, it seemed reasonable that more highly carboxylated peptides would elute from the anion exchange column at higher salt concentrations. As shown below, this behavior was observed and explains the broad range of salt concentrations at which the products elute from the anion exchange column.

Reverse Phase HPLC Purification of Peptides

Prior to mass spectrometry, fractions from the second anion exchange column were combined into groups (Fig. 4), and each group was run on a C18 reverse phase column using a low flow rate elution (data not shown). For each group, a single major peptide peak eluted at essentially the same point late in the gradient. The elution profile of later groups (G5 and G6 in Fig. 4) also contained a moderate size peak just after the FIXQ/S peak. Mass spectrometry of material from this peak revealed a 70510-Da protein that was not investigated further. This reverse phase HPLC step concentrates the peptide and removes both salts and a 280 nm absorbing contaminant. The low flow rate allowed easy rotary evaporation of the samples with minimum peptide loss.

Mass Spectrometry

Even after reverse phase HPLC purification, mass spectrometry on carboxylated material with more than 2 Gla residues could not be done directly. Under the acidic conditions required for positive ion mass spectrometry, peptide precipitation was a problem. Even when precipitates were removed by centrifugation, highly carboxylated FIXQ/S blocked the injection tubing on the mass spectrophotometer. Use of a wide bore PEEK tubing reduced this problem; however, unidentified adducted forms of the peptide dominated the mass distribution. Negative ion mass spectrometry was tried and also produced unidentifiable adducted species.

Our initial attempts at mass spectrometry did reveal that the FIXQ/S starting material included multiple 28-dalton additions. This modification almost certainly represents formyl groups added to the cysteine residues during cyanogen bromide cleavage of the fusion protein and could be reversed by longer treatment with the reducing agent DTT(14) . It should be noted that serine or threonine residues may also be formylated by cyanogen bromide cleavage in formic acid; however, this modification should not be reversible with DTT(16) . A second species of peptide that was 28 daltons less than the observed mass of FIXQ/S was also observed. We have not been able to identify the modification responsible but it appears not to affect carboxylation (see below).

As a solution to the problems mentioned above, we turned to direct mass spectrometry of FIXQ/S as it eluted from a reverse phase microbore column. With this technique, even groups containing highly carboxylated FIXQ/S yielded high resolution data (Fig. 5). As indicated by their presence in the later groups, the higher carboxylation states eluted from the anion exchange HPLC column at higher salt concentrations. All 13 of the possible carboxylation states (0 to 12 added CO(2) groups) are clearly evident. Each carboxylation state has a companion peak which is 28 daltons lighter than itself. As mentioned above, this modification is evident in uncarboxylated FIXQ/S. The even distribution of this form across the carboxylation states suggests that the modification has no effect upon carboxylation. The observed molecular mass of uncarboxylated FIXQ/S was 6954.2 daltons, in good agreement with the expected average mass of 6954.6 daltons. The mass difference between the carboxylation states is 45.3 daltons, corresponding to a specific activity of 40.3 mCi/mmol for the incorporated carbon atom. The added NaH^14CO(3) had a specific activity of 56.4 mCi/mmol, but the presence of cold endogenous bicarbonate in the reaction solutions results in some dilution.


Figure 5: Mass spectroscopic analysis of the groups 1 to 6 clearly shows FIXQ/S at every carboxylation state. The numbers near each peak indicate how many Gla residues would have to be present to yield FIXQ/S molecules of the observed molecular mass. The latter groups which eluted from the anion exchange column at higher salt concentration clearly contain more highly carboxylated FIXQ/S molecules. Prior to mass spectrometry, each group was desalted and purified by reverse phase HPLC. A portion of these purified samples were then loaded onto a C4 reverse phase microbore column and eluted directly into VG Bio Q mass spectrometer to obtain the above profiles. See ``Materials and Methods'' for details.



Quantitation of the average carboxylation state of the product peptides was simplified by the similar ionization efficiencies observed for the various carboxylation states. Under identical mass spectrometry conditions, the relative signal intensities from identical amounts of groups 2 though 6 in Fig. 5were, respectively, 96:100:80:66:51. The fact that the different groups have similar ionization efficiencies argues strongly that the ionization efficiencies of the different carboxylation states within a single group will have similar ionization efficiencies. This allows a reasonably accurate estimate of average carboxylation state of the products by the algorithm given under ``Materials and Methods.'' Because the peak containing highly carboxylated product (G6 in Fig. 5) contains most of the product peptide (Fig. 4), this material dominates the average, which is approximately 7.5 carboxylations per carboxylated product. While an estimate of the possible error in this average is difficult, even unreasonably extreme assumptions about relative ionization efficiencies of similar species within a group would result in a change in the average of less than 1 carboxylation per peptide.

Processive Mechanism

Given that most of the FIXQ/S molecules present in the reaction were uncarboxylated, the relatively high degree of carboxylation among those peptides that were carboxylated suggests a processive mechanism in which many or all of the available glutamates are carboxylated during a single association between the carboxylase and the substrate. A carboxylation mechanism of this type is outlined in Fig. 6A. In a processive mechanism, the rate of release of the product must be slower than the rate of chemical reaction if multiply modified forms are to be produced during a single association between enzyme and substrate. Even if dissociation and reassociation of the partially carboxylated products does occur, it should be minimal prior to accumulation of significant product.


Figure 6: The possible alternative mechanisms that could explain apparent processivity during carboxylation. The carboxylation state of a product is represented by the subscript (e.g. product P(1) is singly carboxylated). In the processive mechanism (A), released products do not reassociate with the carboxylase. In the efficiency-enhanced distributive mechanism (B), binding and release are rapid. In addition, the carboxylation of the first glutamate must be much less efficient than additional carboxylations. Although it is not a necessary feature of either mechanism, the conversion of products to a lower carboxylation state (i.e. the back reaction) has been omitted because it is energetically unlikely and has not been observed.



Efficiency-enhanced Distributive Mechanism

The above data suggest a processive mechanism; however, one other type of mechanism that does not involve processivity could result in apparently processive behavior. If product release is rapid relative to the carboxylation (i.e. the reaction is distributive, not processive), it is still possible for highly carboxylated FIXQ/S to predominate as product, if the carboxylated peptides are dramatically better substrates than the uncarboxylated peptide (Fig. 6B). In other words, if the initial carboxylation event were to result in a large increase in the carboxylation efficiency of the singly carboxylated FIXQ/S as compared to the uncarboxylated FIXQ/S, and if this increase in efficiency were maintained throughout the remaining carboxylation events, then the carboxylated forms could out compete the far more abundant uncarboxylated FIXQ/S. We have termed this special case of a distributive mechanism the ``efficiency-enhanced'' distributive mechanism to reflect the central requirement that the efficiency of modification of the intermediate product forms be enhanced relative to the original substrate. It is important to recognize that the processive and efficiency-enhanced distributive mechanisms are the only way to explain up to 12 carboxylations in FIXQ/S while most substrate molecules remain uncarboxylated.

Direct Competition between Carboxylated and Uncarboxylated FIXQ/S

The most direct way to demonstrate efficiency-enhanced distributive behavior would be to show that partially carboxylated FIXQ/S including the singly carboxylated form is preferentially carboxylated in a reaction containing both carboxylated and uncarboxylated FIXQ/S. However, when a reaction was performed which contained FIXQ/S and the precarboxylated material from group 3 (0 to 2 carboxylations, see Fig. 5), both the uncarboxylated and previously carboxylated peptides were similarly carboxylated (Fig. 7). Quantitation of the carboxylation rate for both uncarboxylated and previously carboxylated peptides is difficult due to low conversion rates and variable backgrounds. Nevertheless, the amount of radiolabeled (precarboxylated) and unradiolabeled (not previously carboxylated) peptide converted to the peak which elutes at the same location as material from group 6 (6 to 12 carboxylations, see Fig. 5) was more than 1% and less than 3% of starting material in each case (Fig. 7). These similar conversion rates indicate that the carboxylation states present have similar carboxylation efficiencies. This result is inconsistent with the substantial enhancement in carboxylation efficiency of the intermediate products required for a distributive mechanism to appear processive.


Figure 7: In direct competition experiments, FIXQ/S with 1 or 2 carboxylations is not a significantly better substrate than FIXQ/S. A 5-ml carboxylation reaction was performed using ^14C-FIXQ/S from group 3 (2.8 µg), 1 µM FIXQ/S (34.6 µg), 12 nM carboxylase, 0.38 mM cold NaHCO(3). Propeptide (proFIX19) was present in the reaction at 0.4 µM. The reaction was initiated by the addition of 111 µM vitamin KH(2), supplemented with an additional 111 µM vitamin KH(2) at 12 h and stopped after 24 h. Products were recovered and separated by anion exchange HPLC (same gradient as Fig. 8). A shows the radioactive profile of ^14C-FIXQ/S from group 3 before (circle) and after (bullet) reaction. Following the reaction, an additional 2.6% of the total label shifted from the elution position of group 3 (1 or 2 carboxylations) to the elution position of the Highly Carboxylated FIXQ/S (6 to 12 carboxylations). B shows the absorbance profile of the postreaction FIXQ/S (upper profile) compared with prereaction peptide (lower profile). The area under the peak labeled Highly Carboxylated FIXQ/S (shown enlarged as an inset) was estimated to be 1.2% of the total peptide absorbance.




Figure 8: Vastly lower concentrations of carboxylated product do not affect the distribution of product carboxylation states. A 200-ml reaction containing 1300 µM FIXQ/S and 0.1 nM carboxylase (200-fold below the carboxylase concentration used in the reaction in Fig. 2) was incubated for 1 h, resulting in the incorporation of 0.4 nM^14CO(2) into FIXQ/S. Despite the fact that at most 0.4 nM FIXQ/S molecules (1 in 3250) were modified, almost all the incorporated radioactivity is found in molecules eluting at the peak that contains product with 6 to 12 carboxylations. Note the absence of an observable product peak in the absorbance profile reflecting the low level of product formation. To dilute the carboxylase without changing the reaction conditions, a solution similar in composition to the buffer containing the purified carboxylase was made (20 mM MOPS, 500 mM NaCl, 20% glycerol, 2 mM EDTA, 0.1% phosphatidylcholine, and 0.7% CHAPS). This was added to the reaction mixture prior to the carboxylase so that the volume of the replacement solution plus the carboxylase continued to be 20% of the total volume. The reaction contained 111 µM vitamin KH(2) and 4 nM proFIX19.



To demonstrate the extent of this inconsistency, we performed a carboxylation reaction with 1.3 µM FIXQ/S and a carboxylase concentration of 0.1 nM. Under these conditions, less than 0.4 nM NaH^14CO(3) was incorporated into the peptide during the course of the reaction; nevertheless, the extent of carboxylation as indicated by the elution pattern of the radiolabeled peptide on anion exchange HPLC (Fig. 8) was essentially the same as observed previously (Fig. 3). For the vastly more abundant uncarboxylated FIXQ/S (1300 nM) to be out competed by the carboxylated forms (below 0.4 nM) for an average of about 7.5 carboxylations would require these forms to be carboxylated more than 3250-fold more efficiently than FIXQ/S. As shown above, this was not the case. In addition, over the range of enzyme concentrations which have been tried (0.1 to 20 nM), the production of the carboxylated forms was proportional to the carboxylase concentration (data not shown). Because the concentration of enzyme affects the concentration of intermediates, this would not in general be the case for an efficiency-enhanced distributive mechanism.

On the other hand, both a similar distribution of carboxylation states and reaction rate proportionality would be expected from a processive enzymatic reaction. The concentration of carboxylase would not affect the distribution of carboxylation states resulting from an association between an enzyme and substrate. In addition, since the reaction velocity expected from a processive mechanism is not affected by the concentration of carboxylated intermediates, the processive mechanism would predict a carboxylation rate proportional to the enzyme concentration when the substrate concentration is essentially constant and not limiting.

A possible difficulty encountered in the above experiments was the presence of propeptide (FIX19) from the purified carboxylase that was used. Fortunately, the presence of propeptide should not affect the experimental outcomes. The concentration of FIXQ/S (1.0 µM) in these experiments was sufficiently above the concentration at half-maximal velocity (0.6 µM), observed in the presence of 0.4 µM propeptide (as in Fig. 7) that most of the carboxylase was bound to FIXQ/S. This observation shows that the carboxylated forms of FIXQ/S are competing primarily with uncarboxylated FIXQ/S rather than with propeptide.

Relative elocities for Carboxylated and Uncarboxylated FIXQ/S

Examination of the efficiency-enhanced distributive mechanism (Fig. 6B) suggests a second independent method for testing carboxylase behavior against this mechanism. The mechanism is a complicated case of a common situation in enzymology: multiple substrates competing for a single enzyme. For the case in which two substrates compete for an enzyme, the relative velocity of conversion of each substrate is given by (17) .

Importantly, the relative reaction velocity is dependent upon the concentrations of both substrates. Extending this simple example to the more complicated situation in our carboxylation reaction, it is still clear that, as the concentration of the carboxylated intermediates increase, the relative rates of carboxylation of uncarboxylated and carboxylated FIXQ/S molecules must change, since the concentration of uncarboxylated FIXQ/S is essentially constant. Similarly, the relative rates of carboxylation of different carboxylation states should change relative to one another.

However, as data in Table 1show, during a time course of carboxylation from 1-22 h, the proportion of carboxylated peptide with about the same anion exchange elution times as Group 3/4 (1-3 carboxylations), Group 5 (3-6 carboxylations), and Group 6 (6-12 carboxylations) were unchanged. Thus, at changing levels of intermediates, additional uncarboxylated FIXQ/S continues to be recruited for carboxylation at the same rate as the carboxylated forms. Once again, this behavior is not consistent with an efficiency-enhanced distributive mechanism; however, it is exactly that expected from a processive mechanism as the concentration of unbound intermediates should have no effect on the distribution of carboxylation states resulting from an association between an enzyme and substrate.




DISCUSSION

For the in vitro carboxylation reaction, only two types of mechanisms could explain how as many as 12 carboxylations occur in carboxylated FIXQ/S molecules, while most substrate molecules remain uncarboxylated. One of these mechanisms, which we have termed the efficiency-enhanced distributive mechanism, is not consistent with the behavior observed for the vitamin K-dependent carboxylase. Neither was partially carboxylated FIXQ/S more efficiently carboxylated than FIXQ/S nor was the reaction velocity and the distribution of the carboxylation states dependent upon the concentration of partially carboxylated FIXQ/S as would be expected for this mechanism.

In contrast, the observed behavior of the carboxylase agrees with that expected from a processive mechanism in which multiple modifications of the substrate molecule occur during a single association between the enzyme and the substrate. In a processive mechanism, the degree of substrate modification should be unaffected by enzyme, substrate, or low partially modified product concentrations, since these variables should not affect the modification process once binding has occurred. In addition, the reaction velocity should be unaffected by low levels of partially modified intermediates and should be linearly proportional to the carboxylase concentration when substrate is not limiting. All of these behaviors were observed during the multiple carboxylation of FIXQ/S by the vitamin K-dependent carboxylase.

A processive carboxylation reaction is also consistent with the size and duration of the burst phenomenon observed during time courses of carboxylation. Given the inaccuracy of the method used to quantitate the carboxylase, the size of the burst is in reasonable agreement with the carboxylase concentration when it is recognized that an average of about 7.5 carboxylations occur during a single association between carboxylase and FIXQ/S. For enzymatic reactions where it has been demonstrated that burst behavior is a reflection of rate-limiting product release, the size of the burst is often the preferred method of determining the concentration of active enzyme. For the carboxylase, this suggests that gel quantitation results in an underestimation of the true carboxylase concentration by a factor of about 2.3-fold.

The processive carboxylation of an average of about 7.5 glutamate residues during a single enzyme/substrate association when combined with an average time per carboxylation per enzyme of 17 min implies that an average substrate molecule is remaining bound to the carboxylase for about 127 min. Thus, the half-time for a substrate to be bound is about 63.5 min, which qualitatively agrees with the time at which the burst is half-completed (see Fig. 2).

For each of the experiments presented, it may be possible to suggest unlikely formal possibilities that could result in the observed behavior. However, taken in total, we believe these experiments demonstrate that the vitamin K-dependent carboxylase is a processive post-translational modification enzyme.

It should be recognized that the partial carboxylation observed in many of the in vitro reaction products is probably due to the unnatural environment of the carboxylase rather than a characteristic of the in vivo mechanism. It is difficult to imagine why processive carboxylation in vivo would not proceed to complete carboxylation of the substrate as complete carboxylation is necessary for the expression of the calcium-dependent, functionally essential domain conformation. Unanswered questions relating to the manner in which the carboxylase recognizes complete carboxylation of its native substrates are central to a full understanding of the mechanism of the carboxylase. This is particularly true as carboxylation does not appear to proceed in a linear sequence along the polymer chain, (^2)making this reaction very different from the linear processivity observed in replication, transcription, and translation(12) .

Interestingly, very few examples of the processive post-translational modification of amino acids have been reported. Most post-translational modifications for which the mechanism is understood involve binding to a particular amino acid and/or recognition site, performing a modification on a single amino acid and releasing the product. There are, however, examples of post-translational modification which are probably processive. Convincing evidence that ubiquitin-protein ligase (18, 19) is processive has been presented. Pulse/chase experiments with analysis during the presteady state period were used to demonstrate multiple ubiquitin additions during a single enzyme/substrate association. Evidence suggests that ubiquitination of histones by ubiquitin carrier protein (19, 20) and phosphorylation of RNA polymerase II by template-associated protein kinase (21, 22) may be processive. These three cases and vitamin K-dependent carboxylation are examples of a type of processivity that results from the association of enzyme and substrate at a binding site distinct from the modifiable residues, followed by modification at these residues.

A second class of processive post-translational modifications may be represented by the hydroxylation of proline by prolyl hydrolase(23) . Prolyl hydrolase has two active sites and modifies protein collagen at a large number of sites. The enzyme probably remains bound to at least one and often two prolyl sites on the substrate for a period of time during which many hydroxylations occur. Coincidentally, other work in our laboratory may fit this method of processivity. The coagulation protease Factor XIa exists as a dimer with two active sites. In the intrinsic blood coagulation pathway, Factor XIa cleaves its substrate Factor IX at two sites generating the active protease Factor IXa. In our laboratory, both cleavages occur without the generation of an intermediate. (^3)An apparent lack of intermediate products is compatible only with a processive mechanism or with the less likely efficiency-enhanced distributive mechanism.

Because processivity is possible for any enzyme which multiply modifies a substrate and which has two binding sites on the substrate, it seems likely that processive post-translational modification is a common phenomenon. Unlike processivity involving nucleic acids, processive post-translational modification will often occur in patterns other than a linear sequence along the polymer chain. This should have profound consequences for the mechanisms of processive post-translational modification enzymes. It appears that the mechanism of vitamin K-dependent carboxylation is an excellent model for studying these enzymes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a postdoctoral fellowship from the American Cancer Society. Current address: Dept. of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016.

Supported by Grants HL06350-30 and HL48318 from the National Institutes of Health. To whom reprint requests should be addressed. Tel.: 919-962-2267; Fax: 919-962-0597.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate; MOPS, 3-(N-morpholino)propanesulfonic acid; vitamin KH(2), vitamin K(1) hydroquinone; proFIX19, AVFLDHENANKILNRPKRY.

(^2)
D. Morris, D. Wright, and D. Stafford, manuscript in preparation.

(^3)
A. Wolberg, unpublished observation.


REFERENCES

  1. Suttie, J. W., and Nelsestuen, G. L. (1980) CRC Crit. Rev. Biochem. 8, 191-223
  2. Suttie, J. W. (1985) Annu. Rev. Biochem. 54, 459-477
  3. Suttie, J. W. (1988) Biofactors 1, 55-60
  4. Furie, B., and Furie, B. C. (1990) Blood 75, 1753-1762 [Medline]
  5. Suttie, J. W. (1993) FASEB J. 7, 445-452 [Medline]
  6. Pan, L. C., and Price, P. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6109-6113 [Medline]
  7. Knobloch, J. E., and Suttie, J. W. (1987) J. Biol. Chem. 262, 15334-15337 [Medline]
  8. Ulrich, M. M. W., Furie, B., Jacobs, M. R., Vermeer, C., and Furie, B. C. (1988) J. Biol. Chem. 263, 9697-9702 [Medline]
  9. Wu, S.-M., Soute, B. A. M., Vermeer, C., and Stafford, D. W. (1990) J. Biol. Chem. 265, 13124-13129 [Medline]
  10. Engelke, J. A., Hale, J. E., Suttie, J. W., and Price, P. A. (1991) Biochim. Biophys. Acta 1078, 31-34
  11. Wu, S.-M., Morris, D. P., and Stafford, D. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2236-2240 [Medline]
  12. Kornberg, A., and Baker, T. A. (1992) DNA Replication , 2nd Ed., Freeman, New York
  13. Wu, S.-M., Cheung, W.-F., Frazier, D., and Stafford, D. W. (1991) Science 254, 1634-1636 [Medline]
  14. Kellaris, K. V., and Ware, D. K. (1989) Biochemistry 28, 3469-3482 [Medline]
  15. Morris, D. P., Soute, B. A. M., Vermeer, C., and Stafford, D. W. (1993) J. Biol. Chem. 268, 8735-8742 [Medline]
  16. Goodlett, D. R., Armstrong, F. B., Creech, J., and Van Breemen, R. B. (1990) Anal. Biochem. 186, 116-120 [Medline]
  17. Ferscht, A. (1985) Enzyme Structure and Mechanisms , 2nd Ed., pp. 111 and 112, Freeman, New York
  18. Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999 [Medline]
  19. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem. 61, 761-807 [Medline]
  20. Haas, A. L., Bright, P. M., and Jackson, V. E. (1988) J. Biol. Chem. 263, 13268-13275 [Medline]
  21. Peterson, S. R., Dvir, A., Anderson, C. W., and Dynan, W. S. (1992) Genes & Dev. 6, 426-438 [Medline]
  22. Dvir, A., Stein, L. Y., Calore, B. L., and Dynan, W. S. (1993) J. Biol. Chem. 268, 10440-10447 [Medline]
  23. De Jong, L., Van Der Kraan, I., and De Waal, A. (1991) Biochim. Biophys. Acta 1079, 103-111

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