Regulation of aldose reductase activity. The influence of effectors on reverse isomerization of the enzyme]

The effects of ligands of active and inhibitory centers of homogeneous aldose reductase from cattle eye lens on glucose reduction were studied. Using spectrophotometric titration and equilibrium gel filtration, the interaction of the enzyme active center with substrates was investigated. It was shown that the reaction kinetics obeys a mechanism with a quasi-equilibrium non-ordered attachment of substrates and isomerization of enzyme complexes with nicotinamide dinucleotide phosphates in the course of the catalytic act. It was found that the NADPH in equilibrium NADP equilibrium in the enzyme active center is shifted to the right; however, NADP dissociation may occur only as a result of the aldehyde reduction. The mechanisms of regulation of the enzyme activity by NADP, ADP and alpha-glycerol phosphate were proposed. It was shown that the binding of catalin and morine to the enzyme results in the inhibition of the enzymatic reaction and in the isomerization blocking. It was found that the inhibitory site of the isomeric form of aldose reductase displays a lower affinity for morine.     

The CXC motif: a functional mimic of protein disulfide isomerase

Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding. Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI. This peptide has a thiol acid dissociation constant (pK(a) = 8.7) that is lower than that of glutathione. These attributes endow the CGC peptide with substantial disulfide isomerization activity. Escherichia coli thioredoxin (Trx) utilizes the active site sequence Cys-Gly-Pro-Cys (CGPC; E degrees ' = -270 mV) to effect disulfide reduction. Removal of the proline residue from the Trx active site yields a CGC active site with a greatly destabilized disulfide bond (E degrees ' >or= -200 mV). The DeltaP34 variant retains high conformational stability and remains a substrate for thioredoxin reductase. In contrast to the reduced form of the wild-type enzyme, the reduced form of DeltaP34 Trx has disulfide isomerization activity, which is 25-fold greater than that of the CGC peptide. Thus, the rational deletion of an active site residue can bestow a new and desirable function upon an enzyme. Moreover, a CXC motif, in both a peptide and a protein, provides functional mimicry of PDI.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Introduction of unnatural amino acids into chalcone isomerase.

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

A highly specific glyoxylate reductase derived from a formate dehydrogenase

A Glu141Asn mutant Paracoccus sp. 12-A formate dehydrogenase catalyzes marked glyoxylate reduction. Additional replacement of the His332-Gln313 pair with His-Glu, which is a consensus acid/base catalyst in D-hydroxyacid dehydrogenases, further improved the catalytic activity of the enzyme as to glyoxylate reduction through enhancement of the hydrogen transfer step in the catalytic process, slightly shifting the optimal pH for the reaction. On the other hand, the replacement induced no marked activity toward other 2-ketoacid substrates, and diminished the enzyme activity as to formate oxidation. Consequently, the formate dehydrogenase was converted to a highly specific and active glyoxylate reductase through only the two amino acid replacements.     

A novel phosphoglucose isomerase (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A resolution

The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.     

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

Oxidative Folding of Conopeptides Modified by <Emphasis Type="Italic">Conus</Emphasis> Protein Disulfide Isomerase

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.     

Chimeric forms of neuronal nitric oxide synthase identify different regions of the reductase domain that are essential for dimerization and activity.

Nitric oxide synthase (NOS) is the enzyme responsible for the conversion of L-arginine to L-citrulline and nitric oxide. Dimerization of the enzyme is an absolute requirement for catalytic activity. Each NOS monomer contains an N-terminal heme-binding domain and a C-terminal reductase domain. It is unclear how the reductase domain is involved in controlling dimerization and whether dimer formation alone controls enzyme activity. Our initial studies demonstrated that no dimerization or activity could be detected when the reductase domain of rat neuronal NOS (nNOS) was expressed either separately or in combination with the heme domain. To further evaluate the reductase domain, a set of expression plasmids was created by replacing the reductase domain of nNOS with other electron-transport proteins, thereby creating nNOS chimeric fusion proteins. The rat nNOS heme domain was linked with either cytochrome P450 reductase, adrenodoxin reductase, or the reductase domain from Bacillus megaterium cytochrome P450, BM-3. All the chimeric enzymes retained the ability to dimerize but were unable to metabolize L-arginine (<8% of wildtype activity levels), indicating that dimerization alone is insufficient to produce an active enzyme. Because the greatest regions of homology between electron-transport proteins are in the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) binding regions, we produced truncation mutants within the nNOS reductase domain to investigate the role of these sequences in the ability of nNOS to dimerize and to metabolize L-arginine. The results demonstrated that the deletion of the final 56 amino acids or the NADPH-binding region had no effect on dimerization but produced an inactive enzyme. However, when the FAD-binding site (located between amino acids 920 and 1161) was deleted, both activity and dimerization were abolished. These results implicate sequences within the FAD-binding site as essential for nNOS dimerization but sequences within amino acids 1373 to 1429 as essential for activity.     

Reaction of triosephosphate isomerase with L-glyceraldehyde 3-phosphate and triose 1,2-enediol 3-phosphate

Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.     

Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase

A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

Isolation and Characterization of the cis-trans-Unsaturated Fatty Acid Isomerase of Pseudomonas oleovorans GPo12

Pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid). We purified the isomerase from the periplasmic fraction of Pseudomonas oleovorans. The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme. N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme. The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids. In contrast to the in vivo cis-trans conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents. Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase. However, when crude membranes from Pseudomonas or Escherichia coli cells were used as phospholipid sources, a cis-trans isomerization was detectable which occurred only in the presence of organic solvents. These results indicate that isolated membranes from Pseudomonas or E. coli cells must contain factors which, activated by the addition of organic solvents, enable and control the cis-trans conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase.     

Structural insight into bi-functional malonyl-CoA reductase

The bi-functional malonyl-CoA reductase is a key enzyme of the 3-hydroxypropionate bi-cycle for bacterial CO₂ fixation, catalysing the reduction of malonyl-CoA to malonate semialdehyde and further reduction to 3-hydroxypropionate. Here, we report the crystal structure and the full-length architecture of malonyl-CoA reductase from Porphyrobacter dokdonensis. The malonyl-CoA reductase monomer of 1230 amino acids consists of four tandemly arranged short-chain dehydrogenases/reductases, with two catalytic and two non-catalytic short-chain dehydrogenases/reductases, and forms a homodimer through paring contact of two malonyl-CoA reductase monomers. The complex structures with its cofactors and substrates revealed that the malonyl-CoA substrate site is formed by the cooperation of two short-chain dehydrogenases/reductases and one novel extra domain, while only one catalytic short-chain dehydrogenase/reductase contributes to the formation of the malonic semialdehyde-binding site. The phylogenetic and structural analyses also suggest that the bacterial bi-functional malonyl-CoA has a structural origin that is completely different from the archaeal mono-functional malonyl-CoA and malonic semialdehyde reductase, and thereby constitute an efficient enzyme.     

Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.     

Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.