Vladimir Leskovac

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of β-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/KB, for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzymes active site were constructed for the free enzyme in the oxidized state, the complex of β-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.

Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase

In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD+....Thr-48....R.CH2OH(H2>O)....Zn++, stretching from His-51 on the surface of enzyme to the active site zinc atom in the substrate-binding site of enzyme. Further, it is based on extensive studies of steady-state kinetic properties of enzyme which were published recently. In this study, we have reported the pH-dependence of dissociation constants for several competitive dead-end inhibitors of yeast enzyme from their binary complexes with enzyme, or their ternary complexes with enzyme and NAD+ or NADH; inhibitors include: pyrazole, acetamide, sodium azide, 2-fluoroethanol, and 2,2,2-trifluorethanol. The unified mechanism describes the structures of four dissociation forms of apoenzyme, two forms of the binary complex E.NAD+, three forms of the ternary complex E.NAD+.alcohol, two forms of the ternary complex E.NADH.aldehyde and three binary complexes E.NADH. Appropriate pKa values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.

Reduction of aryl-nitroso compounds by pyridine and flavin coenzymes

1. A systematic kinetic investigation of the reduction of aryl-nitroso compounds by pyridine and flavin coenzymes and their analogs, in enzymatic and nonenzymatic systems, has been reported. 2. Two main groups of nitroso compounds have been investigated, representatives nitroso-benzene and 1-nitroso-2-naphthol; in all enzymatic and nonenzymatic systems, the former was always reduced to phenyl-hydroxyl-amine and the latter to 1-amino-2-naphthol. 3. Pyridine compounds included NADH, APAD-4H2 and DBNA-4H2 in nonenzymatic systems, and liver alcohol dehydrogenase. Flavin compounds included 1,5-dihydrolumiflavin and various forms of reduced 5-ethyl-lumiflavin, in nonenzymatic systems, and the flavoenzymes glucose-oxidase and NADPH-cytochrome P450 reductase. 5. Pyridine coenzymes and their analogs reduced nitroso compounds by a direct hydride transfer, with a primary kinetic isotope of 9.5 +/- 2.2. 6. All flavin compounds (glucose-oxidase and its nonenzymatic analog 1,5-dihydrolumiflavin and NADPH-cytochrome P450 reductase and its analog 5-ethyl-1,5-dihydrolumiflavin) reduced aryl-nitroso compounds with high efficiency (k2 greater than 10(5)M(-1) min(-1)). 7. The flavin compounds have been shown to be much more efficient reductans of nitroso compounds, compared to pyridine coenzymes, both in enzymatic and nonenzymatic systems; the only exception to this rule presented the extremely efficient reduction of p-substituted aryl-nitroso compounds by liver alcohol dehydrogenase.

A novel substrate for yeast alcohol dehydrogenase – p-nitroso-N,N-dimethylaniline

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the novel reduction of p-nitro-so-N,N-dimethylaniline with NADH as a cofactor. Apparent kinetic constants for this enzymatic reaction are: V2=2.1 s−1, KQ=456 μM, KiQ=119 μM, and KP=1.47 mM, at pH 8.9, 25 °C. This reaction is especially useful for the quantitative determination of NAD+ and NADH by enzymatic cycling.

NAD + binding by yeast alcohol dehydro-genase in the presence of pyrazole and a new method for the determination of the enzyme active site concentration

The pyrazole method of Theorell and Yonetani ( Biochem. Z., 338: 573-553, 1963) has been adapted for the determination of the enzyme active site concentration in yeast alcohol dehydrogenase (EC 1.1.1.1). This method cannot be applied indiscriminately to other alcohol dehydrogenases without modification.

The oxidative part of the glucose-oxidase reaction

1. Kinetic parameters of the oxidative part of glucose-oxidase reaction have been measured with 16 different electron-acceptors and glucose as a substrate. 2. In each case, the rate-limiting portion of the oxidative part of reaction was the formation of the E-FADH2.Acceptor-complex; this rate was pH-independent around the pH-optimum of the enzyme. 3. In each case, E-FADH2 acceptor-complex was undetectable in the steady-state kinetics, with the exception of cytochrome-c. 4. The rates of redox reactions between various forms of reduced 5-ethyl-lumiflavin and five different electron-acceptors have been examined with a conventional spectrophotometry. In each case, it was found that the reactions proceeded at high rates whenever thermodynamically feasible, and were totally prevented in the opposite case. 5. Molecular oxygen was able to oxidize only the neutral form of 5-ethyl-1,5-dihydrolumiflavin to its radical form, at a moderate rate; all other forms of reduced 5-ethyl-lumiflavin were not oxidized by O2. 6. By the comparison of enzymatic and model redox reactions, it was possible to establish the minimal mechanism of the oxidative part of the glucose-oxidase catalytic cycle.

Yeast alcohol dehydrogenase IV. Binding of reduced coenzyme and its fragments

Abstract 1. 1. Commercial preparations of the enzyme are homogeneous on an ion-exchange column chromatography they are heterogeneous with respect to their zinc content, total -SH content, state of oxidation and the reactivity of their -SH groups. 2. 2. Heterogeneous properties of commercial preparations have not influenced their coenzyme binding capacity. All commercial preparations tested in this, and our earlier work (Leskovac & Pavkov-Pericin, 1975; Leskovac et al., 1976), bind approx. 2 molecules of NADH/tetrameric enzyme molecule (mol. wt. 144,000), in a non-cooperative fashion. We have arrived at this binding capacity by 3 different methods: fluorescent titration, gel-filtration and the rate enhancement of the Ellman reaction in the presence of coenzyme. 3. 3. pH-profile of the coenzyme dissociation constant indicates that 2 charged groups on the enzyme, with pK. 7.4 and 9.4, control the enzyme-coenzyme complex formation. 4. 4. Commercial preparations bind weakly coenzyme fragments, adenosyl pyrophosphate ribose or ADP, with a stoichiometry of 4–5 fragment molecules/tetrameric molecule of enzyme. 5. 5. After the binding, the coenzyme or its fragments induce a conformational change in the enzyme; the magnitude of this conformational change decreases in the following order: NADH + acetamide > NADH > adenosyl pyrophosphate ribose > ADP.

NOVEL SUBSTRATES OF YEAST ALCOHOL DEHYDROGENASE - 3, 4-DIMETHYLAMINO-CINNAMALDEHYDE AND CHLOROACETALDEHYDE

4‐Dimethylamino‐trans‐cinnamaldehyde and chloroacetaldehyde are novel substrates of yeast alcohol dehydrogenase (EC 1.1.1.1). In the present work, we have reported the steady‐state kinetic constants for both substrates, and their chemical reactions with the enzyme protein itself. Both substrates are potentially useful for biotechnology, chemoenzyme syntheses and analytical biochemistry.

Novel substrates of yeast alcohol dehydrogenase--4. Allyl alcohol and ethylene glycol.

In the present work, we have determined the steady‐state kinetic constants for yeast alcohol dehydrogenase‐catalyzed oxidation of allyl alcohol (H2C=CH.CH2OH) and ethylene glycol (HOCH2.CH2OH) with NAD+, at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol de‐hydrogenase.

Influence of isoproterenol on net potassium uptake in whole pigeon erythrocytes in vitro

Isoproterenol increases net uptake of potassium in whole pigeon erythrocytes in vitro; effect of 10−5 M isoproterenol is blocked by 10−4 M propranolol. Pentifylline, a potent inhibitor of cAMP-phosphodiesterase, significantly amplifies effect of isoproterenol, indicating that isoproterenol-effect is mediated by cAMP. cAMP alone has no direct influence on net potassium uptake, while dibuturyl-cAMP has a very weak effect. Isoproterenol-effects are also mediated by the cell membrane protein-phosphorylation.