Substrate specificity of acetyl coenzyme A synthetase

Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid. The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis. Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic. The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid. Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy. In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported. The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

A two-step purification of ATP-citrate lyase from rat liver and its use in a fluorometric assay for N-acetylglutamate synthetase

ATP-citrate lyase (EC 4.1.3.8) was purified to homogeneity from the liver of rats maintained on a diet containing no fat and high carbohydrate. The procedure involves two steps: dye-ligand chromatography on yellow MX-6G Sepharose CL-4B and ion-exchange chromatography on DEAE-Trisacryl. The specific activity of the enzyme was 10 mumol X min-1 X mg-1 at 25 degrees C, which is equal to the highest specific activity reported to date. The yield was also the highest reported to date, being in excess of 50%, and the enzyme isolated by this procedure has little proteolytic nicking. The pure enzyme was used to establish a coupled fluorometric assay for N-acetylglutamate synthetase (amino-acid acetyltransferase, EC 2.3.1.1) based on coupling coenzyme A production to the oxidation of NADH via ATP-citrate lyase and malate dehydrogenase. The method is easy to perform compared with existing methods and enables the measurement of 100 pmol X min-1 of N-acetylglutamate synthetase activity. The method is generally applicable for measurement of enzymes which produce coenzyme A. The fluorometric method was used to measure the Km for glutamate and acetyl coenzyme A at pH 7.0 and 25 degrees C, which were 8.2 and 0.4 mM, respectively. Arginine at 1 microM gave half-maximal activation of N-acetylglutamate synthetase.     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Potentiometric determination of coenzyme-dependent oxidoreductase enzymes with recycling of the coenzyme

By enzymatically establishing a rapid (essentially equilibrium) coupling of a redox coenzyme such as NAD with the components of the ferrocyanide–ferricyanide half-cell (e.g., using excess diaphorase) the half-cell potential can be used to monitor another enzymatic reaction involving the same coenzyme. This approach provides a general, rapid potentiometric method of assaying coenzyme-dependent oxidoreductase enzymes. We show that these assay systems can be designed for multiple turnover of coenzyme (in our case NAD) during a single assay thereby amplifying the rate of electromotive force (emf) change with a concomitant increase in sensitivity of enzyme assay. This allows the use of small concentrations of coenzyme and extension of the range of enzyme concentrations that may be assayed.     

Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states

Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the E1 component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pKa for the 4'-aminopyrimidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.     

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.     

A radiochemical assay for mitochondrial acetyl-CoA synthase and octanoyl-CoA synthase from rat heart

A simple radiochemical assay is described for measuring the activity of rat heart mitochondrial acetyl-CoA synthase (EC 6.2.1.1) and octanoyl-CoA synthase (EC 6.2.1.2) using labelled acetate and octanoate. Separation of 14C-labelled reactant from its reaction products was achieved by lyophilization. Enzyme activity was determined by the measurement of incorporation of 14C-labelled short and medium-chain fatty acid into their CoA-derivatives. The method is applicable to the assay of other enzymes where products and substrate may be separated on the basis of the volatilization of one of them during lyophilization.     

A continuous coupled polarographic assay for trehalase.

A continuous, coupled polarographic assay, which couples trehalose hydrolysis to O₂ consumption using glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) as ancillary enzymes has been developed for the measurement of trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity. With this procedure, O₂ consumption was a linear function of time and the coupled reaction rate was directly proportional to the amount of protein assayed with both crude and partially purified enzyme preparations. The limits of sensitivity with this assay correspond to the production of 2.5 nmol of glucose/min. The validity of this assay was confirmed by comparative studies with a discontinuous colorimetric assay for the quantitation of glucose. In addition, the applicability of this assay was appraised by determining the K_m of the enzyme for trehalose. The value obtained with the polarographic assay (i.e., 1.3 ± 0.1 mm trehalose) showed excellent agreement with that obtained using a discontinuous colorimetric method (i.e., 1.2 mm trehalose). Thus the equivalence and applicability studies with the polarographic assay demonstrated that this procedure is a valid and sensitive method for the rapid quantitation of trehalase activity.     

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.     

Characterising Complex Enzyme Reaction Data

The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.     

Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis

The enzymic conversion of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid (i.e. o-succinylbenzoic acid) to 1,4-dihydroxy-2-naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two-step process, was investigated using chirally labelled samples of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate-lyase (EC 4.1.3.1), 2-oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4-(2'-carboxyphenyl)-4-oxobutyrate synthase system and 4-(2'-carboxyphenyl)-4-oxobutyrate: CoA ligase. Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiomeric samples of 2-oxoglutarate enantiotopically labelled at C-3. These samples were converted enzymically to succinate with retention of configuration at C-2 and C-3, and to 4-(2'-carboxyphenyl)-4-oxobutyric acid with retention of configuration at C-3. Isocitrate glyoxylate-lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2-oxoglutarate enantiotopically tritiated at C-4 or at C-3 and C-4. The four variously labelled samples of 2-oxoglutarate were enzymically converted to the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. The resulting variously labelled coenzyme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRe atom of the oxobutyric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C-3 of 1,4-dihydroxy-2-naphthoic acid. The second step in this ring closure reaction is the rate-limiting step.     

Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity, and purified SPT to homogeneity. Unlike eukaryotic enzymes, this enzyme was a water-soluble homodimeric protein. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT showed about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The purified recombinant-SPT showed the characteristic absorption spectrum derived from its coenzyme pyridoxal 5'-phosphate (PLP). The addition of the substrate, L-serine, caused spectral changes indicating the formation of the external aldimine intermediate. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.     

Anchimeric assistance in the intramolecular reaction of glucose-dehydrogenase-polyethylene glycol NAD conjugate

Polyethylene glycol-bound derivatives of NAD(P) (PEG-NAD(P)) are water-soluble macromolecular coenzymes used in continuous enzyme reactors. These NAD(P) derivatives have good coenzyme activity for many dehydrogenases, but some enzymes such as glucose dehydrogenase (EC 1.1.1.47) show very low activity with these derivatives (less than 0.1% of that for native NAD(P)). In this work, we prepared a covalently linked glucose-dehydrogenase-polyethylene glycol-NAD conjugate (GlcDH-PEG-NAD) and found that the conjugate shows a much higher reaction rate than that of the native enzyme plus PEG-NAD: the ratio of the reaction rates of GlcDH-PEG-NAD and the native enzyme plus PEG-NAD is calculated to be 10,000-fold at the concentrations of the enzyme subunit and NAD moiety of 0.31 and 0.65 microM, respectively; the rate of the conjugate is even higher than that of the native enzyme plus native NAD. This rate acceleration is due to the increase in the effective concentration of NAD moiety ("anchimeric assistance") and demonstrates the potential of covalent linking for improving the interaction between an enzyme and a coenzyme derivative.     

Determination of the catalytic activity of cyclomaltodextrin glucanotransferase by maltotriose-methylorange assay

A kinetic assay of cyclomaltodextrin glucanotransferase (EC 2.4.1.19), based on the use of methylorange dye as the indicator of cyclodextrins, was studied and the reaction verified with independent HPLC analyses. The assay was optimized for the enzyme of an alkalophilic Bacillus sp. (ATCC 21783). Described enzymological data provide a reasonable background for detection of possible errors. Numerical correlations with previous assays are presented. This method can be adopted as a standard analysis of any cyclomaltodextrin-forming enzyme.     

A rapid and sensitive radiometric assay procedure for thiamine pyrophosphokinase activity using anion-exchange paper discs

A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent¹ enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable.The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [¹⁴C]thiamine as substrate. After incubation with the enzyme in the presence of Mg²⁺-ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [¹⁴C]thiamine and partial retention of [¹⁴C]TPP. This is quantitatively measured using the liquid scintillation counting technique.A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).     

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.