Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F

Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.     

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.     

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.     

Substrate specificity of Aspergillus oryzae family 3 beta-glucosidase

Among glycoside hydrolases, beta-glucosidase plays a unique role in many physiological and biocatalytical processes that involve the beta-linked O-glycosyl bond of various oligomeric saccharides or glycosides. Structurally, the enzyme can be grouped into glycoside hydrolase family 1 and 3. Although the basic ("retaining, double-displacement") mechanism for the catalysis of family 3 beta-glucosidase has been established, in-depth understanding of its structure-function relationship, particularly the substrate specificity that is of great interest for developing the enzyme as a versatile biocatalyst, remains limited. To further probe the active site, we carried out a comparative study on a family 3 beta-glucosidase from Aspergillus oryzae with substrates and competitive inhibitors of different structures, in attempt to evaluate the site-specific spatial and chemical interactions between a pyranosyl substrate and the enzyme. Our results showed the enzyme having a strict stereochemical requirement (to accommodate beta-d-glucopyranose) for its "-1" active subsite, in contrast to its family 1 counterpart.     

Stereospecific alkylation of cis-3-chloroacrylic acid dehalogenase by (R)-oxirane-2-carboxylate: analysis and mechanistic implications

The enzymes trans-3-chloroacrylic acid dehalogenase (CaaD) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) represent the two major classes of bacterial, isomer-selective 3-chloroacrylic acid dehalogenases. They catalyze the hydrolytic dehalogenation of either trans- or cis-3-haloacrylates to yield malonate semialdehyde, presumably through unstable halohydrin intermediates. In view of a proposed general acid/base mechanism for these enzymes, (R)- and (S)-oxirane-2-carboxylate were investigated as potential irreversible inhibitors. Only cis-CaaD is irreversibly inhibited in a time- and concentration-dependent manner and only by the (R)-enantiomer of oxirane-2-carboxylate. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These findings indicate that the inactivation process involves the initial formation of a reversibly bound enzyme-inhibitor complex at the active site followed by covalent modification. Mass spectral analysis of the inactivated cis-CaaD shows that Pro-1 is the site of modification. It has also been determined that Arg-70 and Arg-73 are required for covalent modification because incubation of either the R70A or R73A mutant with inhibitor does not result in enzyme alkylation. Studies of the pH dependence of the kinetic parameters of wild-type cis-CaaD reveal that a protonated group with a pK(a) of approximately 9.3 is essential for catalysis. The group is likely Pro-1, making it predominately a charged species under the conditions of the inactivation experiments. Two mechanisms could account for these observations. In one mechanism, the oxirane undergoes acid-catalyzed ring opening followed by alkylation of the conjugate base of Pro-1. Alternatively, the oxirane undergoes a nucleophilic substitution reaction where the conjugate base of Pro-1 functions as the nucleophile and an acid catalyst polarizes the carbon oxygen bond. The two arginine residues likely bind the carboxylate group and position the inhibitor in a favorable orientation for the alkylation reaction. These findings set the stage for a crystallographic analysis of the inactived enzyme to delineate further the roles of active site residues in both the inactivation process and the catalytic mechanism.     

Use of activators and inhibitors to define the properties of the active site of normal and Gaucher disease lysosomal beta-glucosidase

Lysosomal beta-glucosidase ('glucocerebrosidase') in peripheral blood lymphocyte and spleen extracts from normal individuals and Ashkenazi-Jewish Gaucher disease type-1 patients were investigated using several modifiers of glucosyl ceramide hydrolysis. The negatively charged lipids, phosphatidylserine and taurocholate, had differential effects on the hydrolytic rates of the normal and Gaucher disease enzymes from either source. With the normal enzyme, either negatively charged lipid (up to 1 mmol/l) increased the reaction rates, while decreasing hydrolytic rates were obtained at greater concentrations. In comparison, the peak activities of the Gaucher enzymes were observed at about 2-3 mmol/l or 5-8 mmol/l of phosphatidylserine or taurocholate, respectively. These negatively charged lipids altered only the velocity of the reactions; the apparent Km values were not affected. Taurocholate or phosphatidylserine also facilitated the interaction of the normal enzyme with conduritol B epoxide, a covalent inhibitor of the catalytic site. Compared to the normal enzyme, the Ashkenazi-Jewish Gaucher type-1 enzyme required about 5-fold greater concentrations of conduritol B epoxide for 50% inhibition. Neutral or cationic acyl-beta-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl beta-glucosides were competitive (or linear-mixed type) inhibitors of the normal splenic or lymphocyte enzyme with competitive inhibition constants (Ki) inversely related to the chain length. With octyl and dodecyl beta-glucoside nearly normal competitive Ki values were obtained with the splenic enzymes from Gaucher patients. These Ki values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-1-O-beta-D-glucoside (glucosyl sphingosine) and its N-hexyl derivative, were noncompetitive inhibitors whose apparent Ki values for the normal enzyme were 30 and 0.25 mumol/l, respectively. The Ki values for these sphingosyl glucosides were about increased 5 times for the Gaucher type-1 enzymes from Ashkenazi-Jewish Gaucher disease type-1 patients. The Ki values of glucosyl sphingosine for the normal or mutant enzymes were directly related to increasing concentrations of phosphatidylserine or taurocholate. This latter site appears to be specifically altered by a mutation in the structural gene for lysosomal beta-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.     

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase.

Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active site geometry of two 1,3-1,4-beta-D-glucan 4-glucanohydrolases. The importance of epoxide stereochemistry for enzyme inactivation

1,3-1,4-beta-D-Glucan 4-glucanohydrolases (EC 3.2.-1.73) from Bacillus subtilis and barley (Hordeum vulgare) with identical substrate specificities but unrelated primary structures have been probed with (R,S)-epoxyalkyl (-propyl, -butyl, -pentyl) beta-cellobiosides and with optically pure (3S)- and (3R)-3,4-cellobiosides as active site-directed inhibitors. The optimal aglycon length for inactivation differs for the two enzymes, and they are differentially inhibited by the pure epoxybutyl beta-cellobioside diastereoisomers. The (3S)-epoxybutyl beta-cellobioside inactivates the B. subtilis enzyme much more efficiently than does the (3R)-isomer, whereas the reverse is true for the barley enzyme. Both enzymes are inactivated by a mixture of the stereoisomers at a rate intermediate of that observed with the individual isomers. The two beta-glucan endohydrolases may therefore employ sterically different mechanisms to achieve glycoside bond hydrolysis in their common substrate. The efficiency and specificity of epoxide-based "suicide" inhibitors may be enhanced significantly by the use of inhibitors bearing only one stereoisomeric form of the epoxide group.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Evidence for an essential arginine in the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a k(inact) of 14.3 +/- 1.1 M(-1) min(-1) in phosphate buffer at pH 7.3 and 30 degrees C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to covalent modification of the enzyme. A possible role for an active site arginine in substrate binding is discussed.     

Purification and properties of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from cells of Aerococcus viridans by a procedure which utilized ammonium sulfate fractionation, DEAE Sepharose CL-6B chromatography, and Sephadex G-100 chromatography. The final preparation was homogeneous by SDS-polyacrylamide gel electrophoresis. The enzyme appears to be a tetramer with a subunit molecular weight of 44,000 and utilizes FMN as a cofactor. The enzyme was highly specific for L-lactate. D-lactate, glycolate, and D,L-2-hydroxybutyrate were not oxidized by the enzyme but were competitive inhibitors. The enzyme could be irreversibly inactivated by incubation with bromopyruvate. This inactivation appears to involve a covalent modification near the active site of the enzyme; however, the flavin cofactor is not the site of this modification.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.     

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.     

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.     

Active-site directed inactivation of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.     

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.     

Green tea polyphenols: novel irreversible inhibitors of dopa decarboxylase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG), and (-)-epigallocatechin (EGC) were found to be inhibitors of Dopa decarboxylase (DDC). EGCG and EGC inactivate the enzyme in both a time- and concentration-dependent manner and exhibit saturation of the rate of inactivation at high concentrations, with efficiency of inactivation values (k(inact)/K(i)) of 868 and 1511 M(-1) min(-1), respectively. In contrast, gallic acid behaves as a weak inhibitor of DDC. Protection against inactivation by EGCG and EGC was observed in the presence of the active site-directed inhibitor D-Dopa. Either EGCG or EGC induce changes in the absorbance and CD bands of the visible spectrum of enzyme-bound PLP. Taken together, these findings indicate the active site nature of the interaction of DDC with both polyphenols. On the basis of the properties of the EGCG-inactivated enzyme, it can be suggested that inactivation could be ascribed to a covalent modification of not yet identified residue(s) of the active site of DDC.     

Suicidal inactivation of the rabbit 15-lipoxygenase by 15S-HpETE is paralleled by covalent modification of active site peptides

Lipoxygenases (LOXs) are multifunctional enzymes that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives; they also convert hydroperoxy fatty acids to epoxy leukotrienes and other secondary products. LOXs undergo suicidal inactivation but the mechanism of this process is still unclear. We investigated the mechanism of suicidal inactivation of the rabbit 15-lipoxygenase by [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid (15-HpETE) and observed covalent modification of the enzyme protein. In contrast, nonlipoxygenase proteins (bovine serum albumin and human gamma-globulin) were not significantly modified. Under the conditions of complete enzyme inactivation we found that 1.3 +/- 0.2 moles (n = 10) of inactivator were bound per mole lipoxygenase, and this value did depend neither on the enzyme/inactivator ratio nor on the duration of the inactivation period. Covalent modification required active enzyme protein and proceeded to a similar extent under aerobic and anaerobic conditions. In contrast, [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE), which is no substrate for epoxy-leukotriene formation, did not inactivate the enzyme and protein labeling was minimal. Separation of proteolytic cleavage peptides (Lys-C endoproteinase digestion) by tricine SDS-PAGE and isoelectric focusing in connection with N-terminal amino acid sequencing revealed covalent modification of several active site peptides. These data suggest that 15-lipoxygenase-catalyzed conversion of (15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid to 14,15-epoxy-leukotriene leads to the formation of reactive intermediate(s), which are covalently linked to the active site. Therefore, this protein modification contributes to suicidal inactivation.