The Importance of Methionine Residues for the Catalysis of the Biotin Enzyme, Transcarboxylase ANALYSIS BY SITE-DIRECTED MUTAGENESIS*

Almost all biotin enzymes contain the conserved tetrapeptide Ala-Met-Bct-Met (Bct, NE-biotinyl-L-ly- sine). In the 1.3 S biotinyl subunit of transcarboxylase (TC), this sequence is present between positions 87 and 90. The conserved nature of these amino acids implies a critical role in the function of biotin enzymes. In order to examine the role of these conserved amino acids, point mutations in the gene encoding the 1.3 S subunit have been made by site-directed mutagenesis to generate A87G, MSSL, MgOL, MSST, MSSC, MSSA, and a double mutant A87M, M88A in the 1.3 S subunit. TC, a multisubunit enzyme containing 12 S, 6 S , and 1.3 S subunits, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate (overall reaction). TC can be dissociated into individual subunits and also reconstituted by assembling isolated sub- units to a fully active form. The mutants of the 1.3 S subunit have been reconstituted with native 6 S and 12 S subunits from Propionibacterium shermanii. The effects of mutations on the activity of TC were com- pared with that of TC-1.3 S wild type (WT) prepared in a similar manner. The results show that any substitution of a residue in the conserved tetrapeptide ratio of the micrograms of protein in the TC complex to the micrograms of biotin, it is possible to calculate the number of 5 subunits attached to the 12 S subunit. The 16 form of TC gives a ratio of 661 which is calculated from the protein content of 16 648 pglnmol and 4 nmol of biotin equal to 0.98 pg (4 X 0.244). Similar calculations have been made for TC complexes with varying amounts of 5 S subunits attached to the 12 S subunit. From these values, a theoretical graph has been drawn which indicates the number of 5 S subunits in various TC-1.3 S complexes based on biotin and protein content.

group from methylmalonyl-CoA to pyruvate (overall reaction). TC can be dissociated into individual subunits and also reconstituted by assembling isolated subunits to a fully active form. The mutants of the 1.3 S subunit have been reconstituted with native 6 S and 12 S subunits from Propionibacterium shermanii. The effects of mutations on the activity of TC were compared with that of TC-1.3 S wild type (WT) prepared in a similar manner. The results show that any substitution of a residue in the conserved tetrapeptide causes impairment of the rate of TC activity. Comparison of gel filtration profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron micrographs of the TC assembled with mutant 1.3 S and with wild type 1.3 S subunits showed that the impairment of the overall activity was not due to a failure of the subunits to assemble into complexes. Steady state kinetic analysis using the mutant 1.3 S subunits indicated that the K , for methylmalonyl-CoA or pyruvate did not change significantly indicating that the binding of substrates is not altered. However, the % , values were significantly lower for mutants at positions 87 and 88 than for those at position 90. The replacement of methionine at position 88 either by hydrophobic or hydrophilic residues significantly altered the activity in the overall reaction, while similar substitution at position 90 did not dramatically alter the kcat. These results suggest that Ala-87 and Met-88 are catalytically critical in the conserved tetrapeptide. Transcarboxylase (methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1)) from propionic acid bacteria (1) is a biotin and metal-containing enzyme (Mr = 1,200,000). The 26 S form of the enzyme consists of a hexameric central 12 S subunit (Mr = 360,000), six dimeric outer 5 S subunits ( M , = 120,000), and 12 biotinyl 1.3 S subunits ( M , = 12,000). The outer 5 S subunits are attached to the central 12 S subunit, three on each face, through biotinyl 1.3 S subunits (2). In addition, 18 S and 16 S forms of transcarboxylase with three and two 5 S subunits attached to the central 12 S subunit, respectively, have been reported (1). The enzyme catalyzes the following reaction which is written as the sum of two partial reactions: The biotinyl 1.3 S subunit serves as a carboxyl carrier between the substrate binding sites on the 12 S (CoA site) and 5 S (ketoacid site) subunits. The biotin is attached to the €-amino group of Lys-89 of the 1.3 S subunit. The biotinyll.3 S subunit contains a highly conserved sequence around the Ne-biotinyl-L-lysine, biocytin (Bct)' (3,4). The tetrapeptide, Ala-Met-Bct-Met, has been conserved in a variety of biotin enzymes throughout evolution. The only reported variations in this sequence occur in chicken and rat acetyl-coA carboxylase where alanine is replaced by valine ( 5 ) and in urea carboxylase where the methionine on the Cterminal side of biocytin is replaced by alanine (4). The tetrapeptide was hypothesized to play a vital role either in biotinylation of Lys-89 by holocarboxylase synthetase, for the removal of biotin by biotinidase, or for the carboxylation/ transcarboxylation through biotin (4). We have previously shown that substitutions at Ala-87, Met-88, or Met-90 by site-directed mutagenesis did not affect biotinylation of the 1.3 S subunit by holocarboxylase synthetase (6). Craft et at.
(7) have shown that biocytin is a much better substrate for biotinidase than the biotinylated peptides, and biotinidase is not active with intact 1.3 S subunit, thus ruling out a requirement of the conserved tetrapeptide in the biotinidase reaction. In this study, we have used site-directed mutagenesis of the 1.3 S subunit to investigate the role of residues in the con-served tetrapeptide in the assembly of TC from its isolated subunits and in the ability of TC to carry out the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate.

EXPERIMENTAL PROCEDURES
Materials-Methylmalonyl-CoA, malate dehydrogenase, reduced nicotinamide adenine dinucleotide, pyruvate, and Rose Bengal were purchased from Sigma. All other chemicals were of reagent grade.
Preparation of Genes and Expression of Mutants of 1.3 S Biotinyl Subunits-Our nomenclature for 1.3 S mutants is illustrated in the following example. When alanine at position 87 was replaced with glycine, it was denoted as A87G, and the TC complex, assembled from such a mutant, is called TC-1.3 S A87G. The construction of 1.3 S subunit mutants 1.3 S M88T, 1.3 S M88L, and 1.3 S M90L has been described previously (6,8). They were derived from the plasmid ptacl.3t which expresses the wild type gene encoding the 123 amino acid 1.3 S subunit in Escherichia coli. Mutants 1.3 S A87G, 1.3 S M88C, 1.3 S M88A, and 1.3 S A87M, M88A were also derived from ptacl.3t using complementary oligonucleotides as cassettes to substitute for the wild type (WT) sequence between an XhoI site at amino acid 85 and a BstXI site at amino acid 89. The complementary oligonucleotides synthesized for the cassettes were (Buffer I) and was dissociated by dialysis against 2 liters of the same buffer. A gentle stream of nitrogen was bubbled continuously to maintain anaerobic conditions. After about 18 h of dialysis, the dialysate, containing dissociated TC, was mixed with solid (NH4)'SO4 a t pH 9.0 to 30% saturation. The resultant precipitate was removed by centrifugation at 24,000 X g for 30 min. The supernatant, containing mostly the biotinyll.3 S subunit and the dimeric outer 5 S subunit was brought to 65% saturation with (NH4)'S04. After centrifugation, the pellet was dispersed in 2 ml of 10 mM ammonium bicarbonate buffer, pH 9.0, containing lo-' M DTT, M reduced glutathione, and 10" M PMSF (Buffer 11) and dialyzed against 1 liter of Buffer I1 for 18 h, with two changes of buffer.
The dialysate from this step was loaded onto a column packed with DE52 ion exchanger (2.5 X 10 cm) (Whatman) which had been previously equilibrated with Buffer 11. The 1.3 S biotinyl subunit was eluted with 80 mM NH,HCO, pH 9.0, containing lo-' M DTT, M reduced glutathione, and M PMSF. After washing with 2 column volumes of 100 mM NH4HC08, pH 9.0, the 5 S subunit was eluted with 300 mM potassium phosphate buffer, pH 6.5, containing 0.1 M DTT, lo-' M reduced glutathione, and M PMSF. A typical yield of 6 mg of 5 S subunit per 100 mg of TC was obtained. On average, the 5 S subunit preparation, when assembled with 12 S and 1.3 S subunits, had an activity ranging from 80 to 110 pmol of oxalacetate formed per min per mg of 5 S subunit.
Protein Determination-The protein content of the fractions containing the TC complex was estimated by the method of Warburg and Christian (11) as described by Layne (12). The protein content of 1.3 S WT and 1.3 S mutants were determined using Rose Bengal according to Elliott and Brewer (13), since the 1.3 S subunit contains only 1 tyrosine and no tryptophan.
Biotin Determination-A modification of the method of Rylatt et al. (14) was used to estimate the amount of biotin in the WT or mutant 1.3 S subunits purified from E. coli. Biotin was quantitated by incubating 2 nmol of 1.3 S WT subunit or mutant subunit in 400 pl of 0.2 M N-ethylmorpholine acetate buffer, pH 8.0, with 50 pg of pronase (Type XIV: bacterial protease, Sigma) at 37 "C for 18 h. The samples were lyophilized after terminating the reaction by boiling at 100 "C for 10 min. The lyophilized sample was dissolved in 0.2 M potassium phosphate buffer, pH 7.2, and the biotin content was estimated by the method of Rylatt et al. (14). For the estimation of biotin in the TC samples, the protein was precipitated with trichloroacetic acid prior to digestion with pronase in order to minimize phosphate interference with the biotin determination. As all preparations of 1.3 S subunits are contaminated with the apo form, the concentration of 1.3 S used in each experiment was calculated on the basis of biotin content and not protein content.
TC Assay-TC was assayed, in the forward direction as previously described (15), by detecting the formation of oxalacetate via a coupled assay system using malate dehydrogenase and measuring the decrease in NADH absorbance spectrophotometrically. The assays for the forward reaction contained the following in a total volume of 0.3 ml: pyruvate, 2.26 pmol; malate dehydrogenase, 4.5 units; NADH, 0.09 pmol; potassium phosphate, pH 6.5,75 pmol; methylmalonyl-CoA, 90 nmol; and the assembled enzyme. Specific activities are expressed as oxalacetate formed in the forward reaction in pmol/min"/mg" of TC.
High Performance Liquid Chromatography (HPLC)-HPLC analyses were performed with a Shimadzu HPLC system equipped with a computer interface and software for the integration and analysis of the peaks in the chromatogram. An ULTROPAC TSK G4000SW (7.5 X 300 mm) gel filtration column was used for subunit separation. The elution of protein was achieved using 0.5 M potassium phosphate buffer, pH 6.5. The protein elution was monitored at 220 nm with a flow rate of 0.5 ml/min.
Electron Microscopy-For electron microscopy, samples were taken up in 0.1 M potassium phosphate buffer, pH 6.5, and cross-linked with glutaraldehyde (0.4%) in order to prevent dissociation during staining with uranyl acetate. The grids for electron microscopy were prepared as described by Wrigley et al. (16). All electron microscopy samples contained 100 pg/ml enzyme, a concentration previously shown to yield the best results. Plates were made of the images at a magnification of ~65,000.
Calculation of the Number of 5 S Subunits Attached to the 12 S Subunit-It is possible to calculate the number of 5 S subunits attached to the 12 S subunit by estimating both biotin and protein concentrations. For example, the 16 S form of the enzyme contains one 12 S subunit, two 5 S subunits, and four 1.3 S subunits (1). From the ratio of the micrograms of protein in the TC complex to the micrograms of biotin, it is possible to calculate the number of 5 S subunits attached to the 12 S subunit. The 16 S form of TC gives a ratio of 661 which is calculated from the protein content of 16 S; 648 pglnmol and 4 nmol of biotin equal to 0.98 pg (4 X 0.244). Similar calculations have been made for TC complexes with varying amounts of 5 S subunits attached to the 12 S subunit. From these values, a theoretical graph has been drawn which indicates the number of 5 S subunits in various TC-1.3 S complexes based on biotin and protein content.
Treatment of Kinetic Data-Initial velocity was measured by varying the concentration of substrates as described by Northrop (17) in a UV 160U Shimadzu spectrophotometer equipped with a temperature control unit. Initial velocity data from each substrate-velocity FITTER, by Leatherbarrow, R.J. from Elseiver-BIOSOFT). The set was analyzed via a nonlinear regression software program (ENZcorrected data were used to construct double-reciprocal plots and were analyzed using weighted least-square regression.

RESULTS
Assembly of Active Enzyme from Its Subunits-TC assembly was achieved by incubating the 1.3 S WT subunit or a 1.3 S mutant subunit with the 12 S and 5 S subunits at 4 "C in 0.5 M potassium phosphate buffer, pH 6.5. It was necessary to maintain the phosphate concentration at 0.5 M in order to achieve assembly. The molar ratio of the 12 S:5 521.3 S biotinyl subunits used for the reconstitution was 1:6:12 unless otherwise stated. This is the stoichiometry of 12 S to 5 S to 1.3 S subunits in 26 S TC which contains the full complement of six 5 S subunits. In separate experiments, it was noted that the activity of the assembled TC was the same if the proportion of 1.3 S subunits used during the assembly was increased from 12 to 18 or 24 (data not shown).
Correlation of Time on Assembly of TC Subunits and Enzyme Activity-The effect of variation of time on assembly and on activity using wild type and mutant 1.3 S subunits is shown in Fig. 1 Fig. 1, no attempt was made to separate the TC complexes from free unassembled subunits. We next developed a technique for separating complexes from free subunits employing HPLC gel filtration chromatography. Under the conditions used (see "Experimental Procedures"), control experiments demonstrate that free 12 S, 5 S, and 1.3 S subunits were separated from the assembled TC (Fig. 2). Chromatographic separation of 12 S subunit showed that in addition to a major peak (number 3 in panel A ) , the preparation was contami-  Table I. nated with a small amount of intact TC (peak number 2 in panel A ) . A mixture of 5 S and 12 S subunits did not interact significantly in the absence of added 1.3 S subunits (panel C ) . The 1.3 S subunit eluted with the salt peak (the column bed volume is 13 ml). After reconstitution with 1.3 S WT and native 12 S and 5 S subunits, TC-1.3 S WT fractionated as a mixture of assembled forms including complexes which fractionated as if they were fully assembled 26 S forms (with six 5 S subunits attached, peak 1 of panel D ) and TC with less than the full complement of 5 S subunits (peak 2 ) (16,18). Isolation of the Complexes Formed from the 12 S, 5 S, and

S W T and S Mutant Subunit and Evaluation of Their
Transcarboxylase Actiuity-Once the conditions were established for the separation of complexes, these were isolated and activities were measured. Activities were determined for peaks 1 and 2 combined as it was not possible to completely separate the 26 S form of TC (peak 1 ) from the partially assembled complexes (peak 2). The results are presented in Tables I and 11. In Table I

Determination of the amount of biotin and estimation of activity per biotin of TC and determination of the number of 5 S subunits in assembled TC
The amount of biotin was determined as described under "Experimental Procedures," and the activity was determined in the forward reaction using assembled TC as described in Table I. A87G showed around 72% and 21% of TC-1.3 S WT activity, respectively, and all other mutants showed very low activities (less than 13%) in the transcarboxylase reaction. A unit of enzyme is defined as the amount of enzyme required to produce 1 pmol of oxalacetate per min at 25 "C. The specific activities in Table I (Table 11) correlated well with the specific activity of the enzyme (Table I) Tables I and I1 would not be similar. The activity correlation in Table I  Subunits-The low activities seen with mutant TC-1.3 S subunits could have been caused by fewer than six 5 S subunits binding to the 12 S subunit. In order to determine whether the low activities were due to a reduced number of 5 S subunits on TC, the number of 5 S subunits per enzyme was determined by two methods. 1. We calculated the number assuming that peaks 1 and 2 from HPLC are made up entirely of TC and free of unbound subunits. The calculations were made as described under "Experimental Procedures.'' The number of 5 S subunits in the TC-1.3 S mutants and TC-1.3 S WT is presented in Table  11. The results indicate that the number of 5 S subunits attached to the 12 S subunit varied from 3 to 6. This 2-fold difference may be partially responsible for the low activity with TC-1.3 S M88T mutant. Although the number of 5 S subunits varied from 3 to 6, the activities per nmol of biotin by themselves were low for TC-1.3 S mutants (Table 11). For example, the TC-1.3 S mutant A87M, M88A contained 12 nmol of biotin per nmol of TC with six 5 S subunits attached to the 12 S subunit as if it were the 26 S form of TC, yet complexes containing this mutant showed only 9-11% activity per mg of protein or nmol of biotin relative to TC-1.3 S WT. In the case of TC-1.3 S M88T mutant, which contained only three 5 S subunits attached to one 12 S subunit, the activity per mg of protein or nmol of biotin did not vary drastically and was 5-6% of TC-1.3 S WT. Since the activities per biotin are approximately the same for various forms of TC, the low activities with various TC-1.3 S mutants were not due to a reduced number of 5 S subunits attached to the 12 S subunit.
2. We experimentally measured the number of 5 S subunits using SDS-polyacrylamide gel electrophoresis analysis. SDSpolyacrylamide gel electrophoresis of TC, TC-1.3 S WT, and TC-1.3 S mutants showed the three bands corresponding to 12 S, 5 S, and 1.3 S before and after the isolation of the complexes. The gels were scanned and the molar ratio of the amount of 12 S, 5 S, and 1.3 S in each TC-1.3 S mutant complex was calculated and compared with that of TC-1.3 S WT (Table 111). The results correlated well with the data in Table I1 in that the estimation of the number of 5 S subunits for every 12 S subunit was as predicted by the calculation method. The results also showed that there were no unusual  complexes like 12 S-1.3 S-12 S or 5 S-1.3 S-5 S because all three subunits were present in the isolated complexes. (These complexes could have been present but not recognized during HPLC gel filtration, possibly because of similarities in molecular weight to that of partially assembled TC complexes.)

Kinetic Parameters of Wild Type and Mutant TC-The K,,,
for methylmalonyl-CoA and pyruvate were determined as described by Northrop (17) for authentic TC from P. shermanii. The K,,, for methylmalonyl-CoA and pyruvate for various TC mutant complexes and for TC-1.3 S WT complex are given in Table IV were 2530 and 260, respectively, which is a 10-fold difference.

DISCUSSION
All biotin-containing carboxylases studied to date have a highly conserved sequence around the biocytin (biotinyl lysine). As shown in Fig. 3, the sequence Ala-Met-Bct-Met is highly conserved throughout evolution except for chicken and rat liver (not shown) acetyl-coA carboxylase, where Ala is replaced by Val (4,5), and urea carboxylase where Met at the C-terminal side of Bct is replaced by Ala (4). Thus, the only two completely conserved amino acids within the tetrapeptide are Met-88 and Bct-90. In this study, we considered the possibility that mutations in the 1.3 S subunit, Ala-87, Met-88, and Met-90 around Biocytin 89, could affect 5 S binding to the 12 S core and/or carboxyl transfer via biotin of the 1.3 S subunit. The multisubunit TC can be readily dissociated and its constituent subunits can be easily separated. This property and the ability to obtain full activity after reconstitution of the isolated subunits has allowed us to manipulate the 1.3 S subunit and to examine the role of the conserved tetrapeptide in TC assembly and activity. Mutations in one of its subunits could affect function in a variety of ways, including holoenzyme assembly, substrate binding, catalytic efficiency of the overall reaction, or catalytic efficiency in either of the partial reactions. Mutations in the tetrapeptide could lead to loss of structure and hence the ability to reassemble TC and to act as a carboxyl carrier. In order to test the competency of the mutants, a reconstitution assay was developed to determine whether mutation at Met-88 and Met-90 had any affect on the activity of TC. The results from Figs. 1 and 2 clearly show that the mutant 1.3 S subunits can promote assembly of TC. The rate of formation of assembled TC from the 1.3 S mutant subunits was comparable to that of TC-1.3 S WT showing that the relative  OADC is oxalacetate decarboxylase, cACC is chicken liver acetyl-coA carboxylase, EcACC is E. coli acetyl-coA carboxylase, UC is urea carboxylase, yPC is yeast pyruvate carboxylase, hPC is human pyruvate carboxylase, sPC is sheep pyruvate carboxylase, aPC is avian pyruvate carboxylase, hPCC is human propionyl-CoA carboxylase, and tbp is tomato biotinyl peptide. Residues identical with the 1.3 S subunit are enclosed in boxes, and residues encircled in a box are not identical. abilities of the mutants to promote assembly has not been changed significantly. The amounts of assembled proteins produced from subunits were also similar for WT and the mutant 1.3 S subunits. As shown in Table I, although some of the mutations in 1.3 S reduced the catalytic activity, the ability to reconstitute was not affected proportionally. The results from rate of assembly, SDS-polyacrylamide gel electrophoresis, HPLC gel filtration, electron microscopy, and protein and biotin content all indicated that mutant 1.3 S subunits and 1.3 S WT can assemble with 5 S and 12 S subunits equally well. These results imply that the residues around biocytin are not involved in binding of 5 S and 12 S subunits and show that the decreased activity of some mutant 1.3 S subunits was not correlated with their ability to promote assembly. We conclude that none of the mutations we have introduced into the tetrapeptide affect the rate or amount of assembled TC from individual subunits although the mutations in the tetrapeptide did affect catalytic function.
Mutations at Ala-87 and Met-88 affected the catalytic activity of TC significantly and to a greater extent than mutation at Met-90. There are, at least, three obvious requirements which must be met if a TC-1.3 S mutant is to have a rate of catalysis comparable to that in TC-1.3 S WT. (i) The 12 S and 5 S subunits must be positioned correctly in the assembled TC by the mutated 1.3 S subunit. If the 12 S and 5 S subunits dissociate because they are not bound together as firmly as in TC-1.3 S WT, the overall reaction will be slowed. (ii) Both partial reactions must be catalyzed by the

Site-directed Mutagenesis
of Transcarboxylase TC-1.3 S mutants at a rate at least as rapid as the overall reaction obtained with TC-1.3 S WT. (iii) The 1.3 S mutant subunit must be positioned in the TC complex to enable carboxyl transfer from the 1.3 S subunit to the substrate sites of the 12 S and 5 S subunits at a rate equivalent to that in TC assembled with 1.3 S WT subunits. Amount of Biotin and Number of 5 S Subunits Present in TC-1.3 S Complexes-We tested the possibility that the number of biotins present in the assembled complexes might be less than that of TC-1.3 S WT, thus explaining the reduced activity in the overall reaction of TC-1.3 S mutants. The data from Table I1 indicate that similar percent activities are obtained for the TC-1.3 S mutants when specific activities are based on either protein or biotin content. Although the number of 5 S subunits attached to the 12 S subunit varied from 3 to 6, this fact did not significantly affect activity. The results clearly indicated that the complexes are formed like in TC-1.3 S WT. The differences in the overall reaction were not due to dissociation of the TC complexes as judged by HPLC gel filtration, thus eliminating requirement (i) as the source of reduced mutant activity.
Kinetic Analysis-The kinetic analysis of 1.3 S mutants indicated that the K, for substrates, methylmalonyl-CoA and pyruvate, were unchanged, while the bat values decreased by as much as 10-fold. The kat/K,,, values indicate a decrease in the catalytic efficiency of the mutated 1.3 S subunits. Ala-87 and Met-88 seem to be more important than Met-90 in the catalytic activity of the enzyme. Met-88 looses activity when i t is substituted either with a hydrophobic amino acid like Leu or Ala or a hydrophilic amino acid like Cys or Thr. A hydrophobic amino acid at position 87 seems to be important for activity because substitution of Gly at this position affected the activity considerably. Even in chicken and rat liver acetyl-coA carboxylase, a Val, another hydrophobic amino acid, is present at this position. It should be noted that mutation at Met-88 did not totally eliminate activity. It is possible that Met-90 might partially assume the function of Met-88 in these circumstances. It is also possible that mutation at Ala-87 or Met-88 may have altered the orientation of the biotin so that it is no longer an effective carboxyl group acceptor. Alternatively, the decarboxylation of carboxybiotin might also be affected in these mutants. Earlier, Kondo et al. (19) showed that the methionines on either side of Bct are important for the carboxylation of biotin. They used synthetic peptides where Met was replaced by Ala to compare the rates of carboxylation of biotin. Their results indicated that the replacement of Met with Ala did not change the K, for biotin in the synthetic peptide but influenced the V, , , of the carboxylation of biotin. They also showed that the Met on either side of Bct decreased the K,,, for biotin in the synthetic peptide when compared to free biotin. They suggested that the cluster of sulfur atoms around biotin may have a role in carboxylation of biotin and that a subtle balance of hydrophobicity and hydrophilicity may be necessary for activity. In our studies, replacement of Met residues around Bct with other amino acids does not fully support this conclusion. The sulfur cluster around biotin may not be important as such, since replacement of Met-90 with Leu did not change activity significantly nor did substitution of Met-88 with Cys protect against activity loss. The substitution of methionines either with hydrophilic or hydrophobic residues gave similar results suggesting that the methionine residue itself at position 88 is important. Further studies are underway to determine whether the mutations at the conserved tetrapeptide affect the carboxylation of biotin of the 1.3 S subunit using isolated subunits in partial reactions 1 and 2.