Robert H. Abeles

Suicide Enzyme Inactivators

I’d like to discuss some of our efforts towards the design of highly specific enzyme inactivators, which can be used in vivo. We hope that such inactivators can be useful as pharmacological agents.

One Protein, Two Enzymes

Two enzymes, designated, E-2 and E-2′, catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2′, overproduced inEscherichia coli from the same gene, have the same protein component. E-2 and E-2′ are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity. E-2′ activity is obtained when Fe2+ is added. Production in intact E. coli of E-2 and E-2′ depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.

A versatile synthesis of peptidyl fluoromethyl ketones

Abstract A versatile synthesis of peptidyl fluoromethyl ketones with potential as serine protease inhibitors is described.

Mechanism of action of cystathionine synthase

Abstract Cystathionine synthase catalyzes the formation of cystathionine from serine, as well as β-chloroalanine, and homocysteine. The stereochemistry of the replacement of the β-OH group of serine by homocysteine was established by converting cystathionine, derived from 2S, 3R-[3-3H]serine, to cysteine with γ-cystathionase. This conversion does not alter the configuration at the β carbon of cystathionine. With previously used procedures ( H. G. Floss, E. Schleicher, and R. Potts (1976) J. Biol. Chem.251, 5478–5483 ) it was determined that the cysteine so obtained was 2R, 3R-[3-3H]cysteine, hence the displacement of the OH group of serine by homocysteine proceeds with retention of configuration. The kinetics of the replacement reaction show an intersecting line pattern. This result, together with the observation that, in the absence of homocysteine, little, if any catalytic conversion of β-chloroalanine to serine occurs, indicates that the β substituent is not released from the enzyme prior to binding of homocysteine. In the absence of homocysteine, the enzyme catalyzes exchange of tritium from [α-3H]serine and β-[α-3H]chloroalanine with solvent protons. At pH 7.8, the rate of this exchange reaction is nearly equal to V of cystathionine formation. The rate of pyruvate formation from serine or β-chloroalanine in the absence of homocysteine is maximally 2% that of the tritium exchange rates. These results show that, in the absence of homocysteine, cystathionine synthase catalyzes α-proton abstraction more rapidly than elimination of the β-substituent. Several mechanisms are proposed by which elimination of the β substituent from the substrate-derived carbanion is prevented in the absence of homocysteine. Cystathionine synthase is irreversibly inactivated by 30 m m 2-amino-4-chlorobutyric acid with t 1 2 = 31 min.

Substrate specificity of nicotinamide methyltransferase isolated from porcine liver

Nicotinamide methyltransferase (EC 2.1.1.1) has been purified over 1300-fold from porcine liver. The enzyme is electrophoretically homogeneous, exhibiting a relative molecular mass of 27,000. In addition to acting on nicotinamide and close structural analogs such as thionicotinamide and 3-acetylpyridine, the enzyme actively accommodates poor analogs such as quinoline, isoquinoline, and 1,2,3,4-tetrahydroisoquinoline as methyl group acceptors. The enzyme may thus have the function of detoxicating numerous alkaloids in vivo. In some cases, the action of the enzyme might paradoxically increase the toxicities of substrates, but the hepatotoxic antibiotic pyrazinamide, which we considered as potentially such an enzyme-activated electrophile, did not function detectably as a substrate for the isolated enzyme.

Inhibition of papain by nitriles: mechanistic studies using NMR and kinetic measurements.

N-(N-acetyl-1-phenylalanyl)aminoacetronitrile is an inhibitor of papain. With 13C NMR spectroscopy we have shown that a reversible covalent adduct is formed with papain. The reversible nature of the covalent-adduct formation was demonstrated with NMR saturation-transfer technique using a DANTE pulse for selective excitation. In addition the covalent adduct was displaced with an aldehyde inhibitor to regenerate the nitrile compound. No hydrolysis of the nitrile was observed. The covalent adduct is most likely a thioimidate formed between the essential thiol and the nitrile. Several p-nitroanilide substrates and their corresponding nitrile inhibitors were examined. A correlation between Ki and kcat/Km was observed. This finding together with the fact that the pH dependence of Ki parallels that of kcat/Km suggests that the interaction of nitriles and papain has considerable transition-state character. In contrast, a nitrile was shown to be an ineffective inhibitor of alpha-chymotrypsin.

The mechanism of inactivation of S-adenosylhomocysteinase by 2′-deoxyadenosine

Abstract S-Adenosylhomocysteinase (SAHase) is irreversibly inactivated by 2′-deoxyadenosine (Hirshfield, M.S. (1979) J. Biol. Chem. 254 , 22–25). In the course of this inactivation, 2′dAd becomes tightly bound to the enzyme, i.e., cannot be removed by gel filtration or dialysis. Inactivation is accompanied by reduction of the enzyme bound NAD. When the inactivated enzyme is denatured, no 2′dAd is recovered. Adenine equivalent to about 80% of the bound 2′dAd is isolated. It is proposed that 2′-deoxyadenosine is first oxidized to 3′-keto-2-deoxyadenosine by enzyme bound NAD. The 3′keto group activates the hydrogen at C-2′ and facilitates elimination of adenine.

17 Disaccharide Phosphorylases

Publisher Summary The term disaccharide phosphorylase is applied to three bacterial enzymes, cellobiose phosphorylase, maltose phosphorylase, and sucrose phosphorylase. Each of the enzymes has been isolated from several sources, and each catalyzes the reversible reaction between the appropriate disaccharide and inorganic phosphate to form glucose 1-phosphate and the respective monosaccharide. All three enzymes can also transfer the glucosyl moiety to acceptors other than phosphate. While considering the mode of action of these enzymes, a decision must be made between two fundamentally different mechanisms. The first mechanism involves the formation of a covalent intermediate (G-E) between the enzyme and the glucosyl moiety of the substrate. The second mechanism, on the other hand, involves no such intermediate. Instead, both substrates initially combine with the enzyme; the displacement reaction then occurs directly without the formation of a covalent intermediate. Sucrose phosphorylase has been purified to near homogeneity from Pseudomonas saccharophila. The purified enzyme appears homogeneous on ultracentrifugation and on starch gel and acrylamide gel electrophoresis. Molecular weight determinations by Sephadex chromatography and from ultracentrifugation data yields a molecular weight of 80,000–100,000. Molecular weight determination by sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis indicated a molecular weight of 50,000, suggesting that the enzyme is composed of two identical subunits.

Enzymatic conversion of the antibiotic metronidazole to an analog of thiamine

We propose that adverse effects of the antibiotic metronidazole may be due, wholly or in part, to its conversion to a thiamine analog and consequent vitamin B1 antagonism. Consistent with this hypothesis, the drug is accepted as a substrate for the thiaminase (EC 2.5.1.2) elaborated as an exoenzyme by the human gut flora constituent Bacillus thiaminolyticus and is also a substrate for the intracellular thiaminase of the mollusk Venus mercenaria. The product, identified as the 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-hydroxyethyl)-2-methyl-4 - nitroimidazolium cation, is a close structural analog of thiamine and is an effective inhibitor of thiamine pyrophosphokinase in vitro. Due to its susceptibility to nucleophilic attack, the analog is unstable, releasing inorganic nitrite under mild conditions. Enzymatic alkylation reactions such as that effected by thiaminase may have general pharmacological significance as a route of increasing the electrophilicity and/or reduction potential of drugs which are heterocyclic weak bases.