Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.     

Substitution of ASP193 to ASN at the active site of ribulose-1,5-bisphosphate carboxylase results in conformational changes.

The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.     

Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl-tRNA synthetase

A highly conserved threonine residue marks the amino acid binding pocket within the editing active site of leucyl-tRNA synthetases (LeuRSs). It is essential to substrate specificity for the Escherichia coli enzyme in that it blocks the cognate leucine amino acid from binding in the hydrolytic editing active site. We combined mutagenesis and computational approaches to elucidate the molecular role of the critical side chain of this threonine residue. Removal of the terminal methyl group of the threonine side chain by replacement with serine yielded a mutant LeuRS that hydrolyzes Leu-tRNA(Leu). Substitution of valine for the conserved threonine conferred similar activities to the wild-type enzyme. However, an additional substitution within the editing active site suggested synergistic interactions with the conserved threonine site that significantly affected amino acid editing. On the basis of our combined biochemical and computational data, we propose that the threonine 252 side chain not only sterically hinders the cognate charged leucine from binding for hydrolysis but also plays a critical role in maintaining an active site geometry that is required for the fidelity of LeuRS.     

Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.     

Crystal structure of the ternary complex of ribulose-1,5-bisphosphate carboxylase, Mg(II), and activator CO2 at 2.3-A resolution

The activated ternary complex, enzyme-CO2-Mg(II), of the dimeric ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum can be prepared in the same crystal form that was used for the crystallographic structure determination of the native nonactivated enzyme (Schneider, G., Br?nden, C.-I., & Lorimer, G. (1986) J. Mol. Biol. 187, 141-143). The three-dimensional structure of the activated enzyme has been determined to a nominal resolution of 2.3 A by protein crystallographic methods. The activator CO2 forms a carbamate with Lys191, located at the bottom of the funnel-shaped active site. In both subunits, this labile adduct is stabilized by a Mg(II) ion, bound to the carbamate and the side chains of Asp193 and Glu194. One solvent molecule was found within the first coordination sphere of the metal ion. The metal-binding site in ribulose-1,5-bisphosphate carboxylase consists thus of at least three protein ligands, all located on loop 2 of the beta/alpha barrel. One additional metal ligand, the side chain of the conserved Asn111, was observed close to the Mg(II) ion in the B-subunit. Other structural differences at the active site between the activated and nonactivated enzyme are limited to side-chain positions. Nevertheless, it is obvious that the hydrogen-bonding pattern in the vicinity of the activator site is completely altered.     

Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity.

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of Km(RuBP) and kcat for Asn111----Gly, the most active mutant, are 420 microM and 0.034 s-1, compared with 13 microM and 3.0 s-1 for wild-type. The mutation of Asn111----Gly causes a more than tenfold decrease in the CO2/O2 specificity factor, tau, tau Asn111----Gly = 0.56 and tau wild-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies.     

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.     

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.     

Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA

The goal of this study was to develop a maltose-producing enzyme using protein engineering and to clarify the relation between the substrate specificity and the structure of the substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). Ala290 at the interface of ThMA dimer in the vicinity of the substrate-binding site was substituted with isoleucine, which may cause a structural change due to its bulky side chain. TLC analysis of the action pattern of the mutant ThMA-A290I, using maltooligosaccharides as substrates, revealed that ThMA-A290I used maltotetraose to produce mostly maltose, while wild-type ThMA produced glucose as well as maltose. The wild-type enzyme eventually hydrolyzed the maltose produced from maltotetraose into glucose, but the mutant enzyme did not. For both enzymes, the cleavage frequency of the glycosidic bond of maltooligosaccharides was the highest at the second bond from the reducing end. The mutant ThMA had a much higher Km value for maltose than the wild-type ThMA. The kinetic parameter, kcat/Km) of ThMA-A290I for maltose was 48 times less than that of wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA were modulated by the residue located at the interface of ThMA dimer near the active site. The conformational rearrangement in the catalytic interface probably led to the change in the substrate binding affinity of the mutant ThMA. Our results provide basic information for the enzymatic preparation of high-maltose syrup.     

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.     

Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 A resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1' substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.     

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.     

Crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate

The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.     

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction

Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolization catalyzed by mutant protein suggests that the major effect of Lys-329 is to facilitate the addition of gaseous substrates (CO2 or O2) to the enediol intermediate. The exchange reaction requires Mg2+, is CO2-dependent, and is inhibited by the transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate. A mutant protein in which Lys-191, the site for carbamylation by CO2 in an obligatory activation step, is replaced by a cysteinyl residue totally lacks proton exchange activity. Barely detectable exchange activity (approximately 0.2% of wild-type) is displayed by the Lys-166----Cys mutant protein, consistent with the previously implicated role of Lys-166 in the deprotonation of ribulose bisphosphate. Retention of exchange activity by the Glu-48----Gln mutant protein, which is slightly active in overall carboxylation, demonstrates that active site Glu-48, like Lys-329, exerts its major effect at some step subsequent to the initial enolization.