RESEARCH
Flavin models probe coenzyme functions Model systems explore dehydrogenation mechanism and interactions of flavins with amino acids ^ ^ Ρ Ι Ι Ι Γ Λ Γ Ω ^ s n o u ^ come as v r U n i t ! I t i l U no surprise that more than a quarter of the papers in the Di vision of Biological Chemistry deal directly or indirectly with enzymes, especially in view of enzymes' impor tance in the functioning and under standing of biological systems. A sub stantial portion of those papers focus on coenzyme function. Highlights of some current coenzyme research include a general mechanism for flavoprotein-catalyzed dehydroge nations and the nature of the associa tions between flavin coenzymes and amino acid residues of flavoproteins, information that is required to under stand flavin coenzyme function. Model. Although there are an enor mous number of oxidation-reduction enzymes that require one of the fla vin coenzymes—flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)—the mechanisms of most of these reactions are not well under stood. One reason is that the flavoprotein dehydrogenases vary among them selves according to the nature of the donors and acceptors, the number of flavin molecules per enzyme and per active site, and the accessory compo nents present such as nonheme iron, heme, or molybdenum. Dr. Gordon A. Hamilton of Pennsyl vania State University, University Park, and graduate student Lawrence E. Brown are studying model systems for some enzymic reactions in which FMN and FAD are involved. They are attempting to establish a general mode of action of the flavoenzymecatalyzed dehydrogenations.
In many enzymic reactions involving FMN and FAD, Dr. Hamilton ex plains, one step in the sequence in volves transfer of hydrogen from an alcohol or an amine to-the oxidized flavin to give the reduced flavin. Dr. Hamilton and Mr. Brown have ob served for the first time the dehydro genation of certain alcohols and amines by a flavin derivative in a nonenzymic system under anaerobic conditions in the dark. Spectrum. Specifically when 10phenylisoalloxazine, a flavin derivative, is reacted with methyl mandelate and potassium terf-butoxide in anhydrous dimethylformamide tert-butyl alcohol under anaerobic conditions, the re search workers observe a spectrum that is typical of the reduced fla vin derivative. Upon addition of oxy gen the spectrum indicates that the oxidized form of the flavin is regen erated in more than 90c/c yield. If reduction of 10-phenylisoalloxazine to its reduced form is due to de hydrogenation of methyl mandelate, Dr. Hamilton says, methyl phenylglyoxylate would be an expected product. In the analysis process methyl phenylglyoxylate is hydrolyzed to its acid. The chemists do, in fact, find this acid in the expected amount on workup of the reaction mixture. When they re place methyl mandelate with the methyl ester of phenylglycine or the diethyl ester of aminomalonate they also obtain a similar reduction of 10phenylisoalloxazine and oxidation of the substrate. Also they observe an oxygen-reversible reduction of lumiflavin, another flavin derivative, by
using any of the three substrates under similar conditions. The Penn State chemists conclude that only alcohols and amines that have a relatively acidic alpha hydrogen —for example, esters of α-hydroxyl and α-amino phenylacetic acids—are readily dehydrogenated. Acylated or ether derivatives of these compounds don't reduce flavin under their reaction con ditions. And the chemists observe oxygen-reversible reduction of the fla vin only with a strong base in an or ganic solvent. As a result of studies with their model systems, Dr. Hamilton and Mr. Brown propose a general mechanism for the dehydrogenation step in flavin reactions. They believe that both hy drogens are transferred as protons. In order to maintain electrical neutrality in the transfer, they propose that an intermediate covalent compound be tween the oxidant (flavin ring system) and reductant is formed; the formation and breakdown of this intermediate provide a mechanism for electron transfer. The covalent compound would be formed, Dr. Hamilton says, by the substrate reacting at an electrophilic site of the flavin ring, prob ably position 4a (see diagram). The distinctive feature of the mech anism proposed for flavoenzyme-catalyzed dehydrogenations is that both hydrogens are transferred as protons, Dr. Hamilton emphasizes. Thus the mechanism is closely related to the mechanism for most other nonredox enzymic reactions. In particular gen eral acid and/or general base catalysis by suitably placed groups on the en-
.Dehydrogenation may proceed through covalent intermediate
flavin derivative (oxidized form)
32 C&EN SEPT. 28, 1970
ahrelitii substrate îEfizytne examples: gteoose oxidase, factate dehydrogenase, choline deHydrogenase)
\ H Intermediate covalent compound
Fîayift derivative (reduced form)
zyme surface would be expected to increase the rate of the reaction. This may be one of the principal ways in which the enzyme increases the rate relative to that of a model system, he says. Further, Dr. Hamilton believes that this general type of mechanism is applicable to many other enzymic redox reactions as well, not only those involving flavin coenzymes. Interactions. Perhaps one of the first prerequisites, however, to understanding how flavin coenzymes function is determination of the site and nature of flavin coenzyme binding to amino acid residues within the holoenzyme. Cornell University chemist Donald B. McCormick and his coworkers are currently studying the nature of the nonionic association between flavins and amino acid residues in flavoprotein enzymes. The importance of strong ionic interactions between anionic flavin coenzymes and the specific cationic sites in proteins with which they function as flavoproteins has long been recognized by biochemists, Dr. McCormick says. But in addition to ionic associations, the binding and oxidation-reduction function of flavin coenzymes involves the association of the isoalloxazine ring system of such flavin coenzymes as FAD. Dr. McCormick, like Dr. Hamilton, deals largely with model systems in which he studies synthetic flavin derivatives in order to get a handle on what is actually occurring within the catalytically functional enzyme. Dr. McCormick and his colleagues, Dr. W. Fory, Dr. R. E. MacKenzie, and Dr. S. Y.-H. Wu, have investigated the properties of model flavinyl peptides in solution in inter- and intramolecular association with aromatic amino acid residues such as tyrosine and tryptophan. Using physical techniques including measurements of light absorption and emission, infrared spectroscopy, and proton magnetic resonance, they have delineated many of the features of such interactionsfirst in their model synthetic flavinyl peptides and more recently in naturally occurring flavoproteins. They find that in more aqueous environments the flavin association with the aromatic amino acids is mainly through coplanar stacking with much of the benzenoid portion of the flavin shielded. As the environment becomes more nonpolar the pyrimidinoid part of the flavin ring system increasingly participates through hydrogen bonding to amino acids such as tyrosine or tryptophan. For example, quite recently Dr. McCormick, Dr. Wu, Dr. C.-W. Wu, and S. C. Tu have observed that the fluorescence of a given apoenzyme can be quenched and/or shifted upon binding a flavin coenzyme. The blue shift ob-
Cornell's Dr. McCormick studies nonionic interactions in flavinyl peptides
served upon reconstitution of D-amino acid apooxidase with FAD indicates a likely interaction whereby tryptophan residues become more masked in a nonpolar environment. From the model systems it is only a step to observing the same interactions in naturally occurring systems, Dr. McCormick asserts. This is important because the associations of flavin
coenzymes with particular amino acid residues of proteins markedly affect the redox behavior of the two-partner system and confer stereochemical specificity for the substrate, he adds. Finally, an understanding of flavinamino acid interaction is becoming helpful in photochemical studies aimed at elucidating certain of the active-site residues in flavoproteins.
Nuclear decay rates assist chemical rate measurements Chemical nuclear rate coupling can be a big help in studying the chemical dynamics of fast chemical processes by various experimental techniques, such as crossed molecular beams. The method consists of using nuclear decay rates to measure chemical rates under controlled conditions. In describing the concept to the Division of Nuclear Chemistry and Technology, Dr. J. Robb Grover of Brookhaven National Laboratory emphasized that the technique differs from radioactive tracer applications, which make use of radioactive decay only for efficient detection. It differs also from hot atom chemistry, which is concerned only with energetic species that result from the dynamics of nuclear transitions. Chemical nuclear rate coupling makes use of nuclear decay rates by incorporating a short-lived radioactive nuclide in the reaction. The main idea, Dr. Grover says, is to arrange apparatus so that the observed signal depends on time-dependent processes, such as translation and rotation, of the reaction under study.
^CHICAGO
The nuclear technique is unlike many detection methods, such as those using short light pulses for detecting product species following a sudden disturbance. In these, signal-to-noise ratio is dependent on resolution time. With the nuclear method, the ratio is independent of time, since at steady state, birth rate of nuclei equals decay rate, and the signal is thus independent of the mean life of the nuclei. High. This independence and the strong suppression of noise because of rapid nuclear decay lead to one of the method's main advantages, a high signal-to-noise ratio. The technique can be widely applicable. Dr. Grover notes that there are more than 130 nuclides of 69 elements available with half-lives between 1 _ 1 and 10~5 second. A prototype experiment being carried out by Dr. Grover and coworkers is a good example. In the experiment, a molecular beam of HAt made with astatine-217 (0.032-second half-life) is crossed with a beam of bromine atoms, with detection of reaction product astatine and scattered HAt being carried out with chemically selective detectors. SEPT. 28, 1970 C&EN 33
