A sex-limited serum protein variant in the mouse: Hormonal control of phenotypic expression

The sex-limited protein (Slp) antigen of the mouse is first detected in the serum of strain DBA/2J males at 5–6 weeks of age and reaches full adult levels by 10 weeks. This antigen is normally absent in females. Immature DBA/2J males castrated at 3 1/2 weeks of age failed to develop Slp antigen, while DBA/2J females treated with testosterone propionate starting at 3 1/2 weeks developed normal adult male levels of Slp antigen. Similar hormone-influenced effects were demonstrated in adult males and females of the same strain. Experiments indicated that testosterone does not act directly in the serum to expose Slp antigenic sites. Testosterone treatment of both males and females of strain C57BL/10JSf, which does not carry the gene for the presence of the Slp antigen, failed to stimulate the appearance of the antigen. Thus, the presence of Slp antigen in the serum is dependent on both the proper genotype and the presence of male hormone.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.), and U.S.P.H.S. Career Development Award K3-HE-24,980 (D.C.S.).     

Purification, identification and properties of cysteine cathepsins from animal tissues

The paper deals with the modern methods of purification of cathepsins B, H, L, N, S. The ways to control their separation in the course of purification are discussed. The multiplicity of molecular forms of these enzymes is analyzed.     

Two histone H1-specific protein-lysine N-methyltransferases from Euglena gracilis. Purification and characterization

Two forms of a histone H1-specific S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III) have been purified from Euglena gracilis 48- and 214-fold, respectively, with yields of 3.4 and 4.6%. The enzymes were purified on DEAE-cellulose and histone-Sepharose affinity chromatography and found to be highly specific toward histone H1 as a substrate. However, one of the enzymes also methylates other histone subfractions to a limited extent. Of the proteins other than histones, only myosin showed measurable methyl-accepting capability. Both enzymes were found to be inhibited by S-adenosylhomocysteine (D and L forms), S-adenosyl-L-ethionine, and sinefungin. While the Ki values for S-adenosyl-L-ethionine were similar for both enzymes, the values for S-adenosyl-L-homocysteine and sinefungin were 10-fold lower for the second form. The Km values for histone H1 and S-adenosyl-L-methionine were found to be 3.1 X 10(-7) and 2.7 X 10(-5) M, respectively, for the first enzyme, and 4.4 X 10(-7) and 3.45 X 10(-5) M for the second. Peptide analysis of methyl-14C-labeled H1 revealed that the two enzymes methylate different sites within the histone H1 molecule. The two enzymes were found to have molecular weights of 55,000 and 34,000, respectively. Both enzymes have an optimum pH of 9.0, which is identical to that of other protein-lysine N-methyltransferases thus far identified.     

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.     

Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding

Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).     

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.     

Genetic and biochemical studies of the adrenal lipid depletion phenotype in mice

Study of F₁ and backcross progenies from crosses between DBA/2 and strains carrying the adrenal lipid depletion (ald) gene indicated that the adrenal lipid depletion character is due to the operation of two or more interacting gene loci. In C57BL/10, DBA/2, and their F₁ hybrids, variation in adrenocortical sudanophilia was highly correlated with both cholesterol ester content and the capacity for the production of corticosterone in vitro. In F₁×DBA/2 backcross progeny, however, the degree of adrenocortical sudanophilia remained correlated only with cholesterol ester content and not with the capacity for corticosterone production. Thus, in these strains, adrenal cholesterol ester concentration is not necessarily the determining factor of corticosterone production in vitro; the two phenotypic characters are controlled by independent genetic mechanisms.This work was supported by MH 10976 and HD 00801 (U.S.P.H.S.). J.G.M.S. is a recipient of a Wellcome Foundation Travel Grant. R.B.C. is a recipient of U.S.P.H.S. Research Scientist Award K5-47, 413 of the National Institutes of Mental Health.     

Demonstration of an intercellular substance in mouse cerebral cortex

Summary Specimens of mouse cerebral cortex were preserved by freezing and drying and treated in vacuo with osmium tetroxide vapors. In the neuropil of the molecular layer dense, osmiophilic laminae, presumably plasma membranes of immediately adjacent cell processes, were found to be separated by an intercellular space, which appeared relatively electron lucent in unstained sections. In sections stained with uranyl acetate an intercellular substance was demonstrated, the visualized density of which was reduced when staining was accomplished at low pH. These reactions suggest the presence of a non-lipid, anionic intercellular substance which may contain acid mucopolysaccharide.This work was supported by U.S.P.H.S. Research Grants NB 05175, NB 07044 and Career Development Award 1K3 NB 4929.     

PectinaseAspergillus sp. polygalacturonase: Multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases

Nine forms ofAspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with freea-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from otherAspergillus species (72–75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66–71% residue differences toward theErwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties     

PectinaseAspergillus sp. polygalacturonase: Multiplicity,divergence, and structural patterns linking fungal,bacterial, and plant polygalacturonases

Nine forms ofAspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with freea-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from otherAspergillus species (72–75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66–71% residue differences toward theErwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties     

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.     

Magnetization studies of the active and fluoride-inhibited derivatives of the reduced catalase of Lactobacillus plantarum: toward a general picture of the anion-inhibited and active forms of the reduced dimanganese catalases

The magnetic properties of the reduced catalase from Lactobacillus plantarum have been studied for the active enzyme and its fluoride complex through variable field/variable temperature magnetization measurements. The magnetic exchange interaction deduced from these experiments [fluoride complex: - J=1.3(1) cm(-1); active enzyme: - J=5.6(5) cm(-1); H=-2 J S(1) S(2)] are similar to those presently obtained in a re-analysis of the data for the corresponding forms of the Thermus thermophilus enzyme (previously published in 1997, Angew Chem Int Ed Engl 36:1626-1628): phosphate complex: - J=2.1(2) cm(-1); active enzyme - J=5.0(3) cm(-1). These results concur to a unified picture for the two enzymes, consistent with the presence of a hydroxide bridge in the reduced active catalases and its replacement by an aqua bridge in the anion-inhibited enzymes as the main mediators of the magnetic exchange.     

Genetic variation of the cytoplasmic and mitochondrial malic enzymes in the monkey Macaca nemestrina

Electrophoretic variation of both the cytoplasmic and mitochondrial forms of the malic enzyme is described in Macaca nemestrina. Pedigree analysis of the observed phenotypes demonstrates that the two subcellular forms of the malic enzyme are genetically independent. The identity of the electrophoretic phenotypes in brain, heart muscle, liver, kidney, adrenal, and spleen from any given individual shows that each subcellular form is determined by the same genetic locus in a wide variety of tissues. After separation by ion exchange chromatography, the cytoplasmic and mitochondrial malic enzymes were shown to be distinct in their heat stability and K _m for malate, but no significant differences were found among the variants of the cytoplasmic enzyme or among the variants of the mitochondrial enzyme. It is possible that the polymorphism of the mitochondrial malic enzyme is selectively neutral.This study was supported by grant GM-15253 from the National Institutes of Health. One of us (G.S.O.) was a Special Fellow, U.S. Public Health Service (5F3 HD 43, 122-02); Fellow, National Genetics Foundation.     

Studies of homogeneous "biosynthetic" L-threonine dehydratase from Escherichia coli K-12. Some kinetic properties and molecular multiplicity.

"Biosynthetic" L-threonine dehydratase (EC 4.2.1.16) was purified to a homogeneous state with 29% yield of total activity from Escherichia coli K-12. The homogeneity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecyl sulphate. The enzyme consisted of equal subunits having a molecular weight of about 57 000. The polyacrylamide gel disc electrophoresis has shown that the native enzyme consisted of a set of oligomeric forms. The multiplicity of molecular organization of the enzyme was reflected in complicated kinetic behaviour: at pH greater than 9 on the plots of initial reaction rate (v) versus initial substrate concentration ([S]o) there were four inflexion points (two intermediate plateaux), the position and deepness of which depended on enzyme concentration. At pH 8.3 on the v versus [S]o plots appeared two inflexion points (one intermediate plateu), the position of which practically did not depend on enzyme concentration in the reaction mixture, but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms, isolated by the first electrophoresis, has shown that the oligomeric forms underwent a slow polymerization. It was suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms, having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of v versus [S]o plots. The rate of equilibrium installation between the oligomeric forms was small in comparison with the enzyme reaction velocity, that lead to the complex kinetic curves, appearing as a result of summing up of the kinetics inherent to theindividual forms.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Purification ofPseudomonas fluorescens subsp.cellulosa endoglucanases produced inEscherichia coli

Both plasmid pPFC4, which contains 10.6 kb, and a derivative of pPFC4—viz., pPFC4-4.6—which contains 4.6 kb ofPseudomonas fluorescens subsp.cellulosa DNA, direct the synthesis of six distinct endoglucanases inEscherichia coli. Two of these enzymes were purified to homogeneity in a single step by means of anion exchange HPLC. One enzyme has a molecular weight of 30.0 ± 1.0 kDa, an isoelectric point of 7.5, and a specific activity of 3470 U of activity/mg of protein, whereas the other has a molecular weight of 38.5 ± 1.0 kDa, an isoelectric point of 6.7, and a specific activity of 18,050 U of activity/mg of protein. On the basis of the amino acid composition, the 38.5 kDa enzyme appears to be a modified version of the 30.0 kDa enzyme. Thus, the multiplicity of endoglucanases produced inE. coli/pPFC4-4.6 cells may be owing to the posttranslational modification of a smaller number of primary translation products.     

Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina

An examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] shows the presence of four, bis-His-ligated, c-type hemes and one flavin adenine dinucleotide, FAD. The heme groups provide a "molecular wire" for the delivery of electrons to the FAD. Heme IV is closest to the FAD (7.4 A from heme methyl to FAD C7), and His61, a ligand to heme IV, is also close (8.4 A to FAD C7). Electron delivery to the FAD from the heme groups must proceed via heme IV, as hemes I-III are too far from the FAD for feasible electron transfer. To examine the importance of heme IV and its ligation for enzyme function, we have substituted His61 with both methionine and alanine. Here we describe the crystallographic, kinetic, and electrochemical characterization of the H61M and H61A mutant forms of the Shewanella fumarate reductase. The crystal structures of these mutant forms of the enzyme have been determined to 2.1 and 2.2 A resolution, respectively. Substitution of His61 with alanine results in heme IV having only one protein ligand (His86), the sixth coordination position being occupied by an acetate ion derived from the crystal cryoprotectant solution. In the structure of the H61M enzyme, Met61 is found not to ligate the heme iron, a role that is taken by a water molecule. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the H61M-Fcc(3) and H61A-Fcc(3) mutant enzymes are catalytically active but exhibit marked decreases in the value of k(cat) for fumarate reduction with respect to that of the wild type (5- and 10-fold lower, respectively). There is also a significant shift in the pK(a) values for the mutant enzymes, from 7.5 for the wild type to 8.26 for H61M and 9.29 for H61A. The fumarate reductase activity of both mutant enzymes can be recovered to approximately 80% of that seen for the wild type by the addition of exogenous imidazole. In the case of H61A, recovery of activity is also accompanied by a shift of the pK(a) from 9.29 to 7.46 (close, and within experimental error, to that for the wild type). Pre-steady-state kinetic measurements show clearly that rate constants for the fumarate dependent reoxidation of the heme groups are adversely affected by the mutations. The solvent isotope effect for fumarate reduction in the wild-type enzyme has a value of 8.0, indicating that proton delivery is substantially rate limiting. This value falls to 5.6 and 2.2 for the H61M and H61A mutants, respectively, indicating that electron transfer, rather than proton transfer, is becoming more rate-limiting in the mutant enzymes.     

Structural basis for the inactivity of human blood group O2 glycosyltransferase

The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fuc alpha1-2Gal beta-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O(2) enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O(2) blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.