Morton J. Gibian

The oxidation of mercaptans by flavins

The reactions of flavins (isoalloxazines) with organic molecules are of interest as possible model systems for the catalytic reactions of vitamin B2 (riboflavin) requiring proteins. The flavoenzyme lipoic acid oxidase catalyzes the oxidation of dihydrolipoic acid (6,8-dimercaptoctanoic acid) to lipoic acid, 3-(6-valeryl)-1,2_dithiolane, and other flavoenzyme systems perform thiol to disulfide oxidations either as the primary reaction or as auxiliary processes to the main catalytic reactions(l,2). GascOigne and Radda(3) reported that dihydrolipoic acid reacts with flavins in the absence of enzyme to produce lipoic acid and reduced flavin. It was noted that the reaction was first order in both flavin and in dihydrolipoic acid, and that it was also base catalyzed (the pH and buffer dependencies were obviously rather complicated). On the basis of this information and with the additional evidence that the relative rates for different flavins followed the same pattern as did the half-wave potentials for the two-electron polarographic reduction of the substituted flavins, the authors concluded that a two electron transfer occurs between the thiolate anion of dihydrolipoic acid and flavin to directly produce lipoic acid and reduced flavin. As part of our interest in flavin redox reactions(4) we wish to report our preliminary results of a survey of the reaction between flavins and mercaptans to produce dihydroflavins and disulfides according to eq 1.

The oxidation of organic anions by flavins

Abstract The reduction of flavins by α-hydroxyketone enolates leads to dihydroflavins and diketones. Isoalloxazines related to flavins (good analogs for their redox chemistry) are reduced by 9,10-dihydroanthracene and succinonitrile carbanions and by propiophenone enolate (in the latter case to give the isoalloxazine radical anion). Alkali metal alcoholates, with an appropriate isoalloxazine, produce 50% yields of isoalloxazine radical ion, presumably via a substitution and electron transfer process. The ease of carbanion (and complete lack of conjugate acid) oxidation suggests a general mode of flavin redox function.

The formation and activities of substituted phenacylchymotrypsins

Abstract 1. 1.|The preparation of derivatives of α-chymotrypsin with p -nitrophenacyl bromide, phenacyl bromide and p -methoxyphenacyl bromide is described. 2. 2.|For the p -nitrophenacyl derivative, the new 350-nm absorption is pH independent from pH 2.88 to 11.25, mitigating against a major change in environment at the new chromophore over this range. 3. 3.|The enzyme derivatives (likely at Met-192) show different decreased but definite catalytic activities towards substrates of α-chymotrypsin. In each case, this activity is due to the modified enzyme itself. 4. 4.|Phenacyl- and p -methoxyphenacylchymotrypsin show generally increased dissociation constants and decreased catalytic constants. Below pH 8.5, p -nitrophenacylchymotrypsin towards ethyl -N- acetyl - l - tryptophanate has only decreased k cat values compared to native enzyme, K m being unaltered. The pH dependencies of k cat below pH 8.5 and K m over the entire range are identical for parent and nitrophenacyl enzyms. Above pH 8.5, k cat for the enzyme derivative increases and is still rising at pH 10, having reached over twice its value at pH 8.5. The effect is reversible. 5. 5.|These results are taken to indicate that the conformational change occurring in α-chymotrypsin at pK 9 occurs for the nitrophenacyl enzyme as well, but, in the latter case, it reverses the original inhibition of k cat by the nitrophenacyl group.

Oxidation of pyruvic acid by flavins in the presence of amines

Abstract Pyruvic acid or other enolizable α-keto acids in the presence of primary or cyclic secondary amines in aprotic polar solvents efficiently reduce flavins and isoalloxazines to their 1,5-dihydro derivatives. The other product of this reaction is the diamide of citraconic acid (1). In the absence of flavin, the diamide of methylsuccinic acid (2) is obtained as the major product. The specificity for α-keto acid and amine, stoichiometric requirements, rates and quantities of CO2 evolution, and the kinetics of the reaction were studied. On the basis of these data a mechanism is proposed that involves an enamine condensation followed by decarboxylation and then either reaction with flavin to give ultimately (1) and dihydroflavin, or reaction with an additional amine to give (2). The possible import of this kind of mechanism in biochemical systems is briefly discussed.