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Biomedical subjects

A L Fink

Publications and source records attributed to A L Fink.

At least 127 records · Page 7Linked to original sources
PubMed1980

The binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures. A three-step reaction.

The binding reaction of NADH to liver alcohol dehydrogenase was investigated at temperatures to -45 degrees C using a cryosolvent composed of 50% aqueous dimethylsulfoxide. At temperatures below -30 degrees C, the reaction produced biphasic changes in the protein fluorescence. The slow phase caused a 15 to 20% quenching of protein fluorescence, reached a maximum at 50 to 100 micro M NADH, and had an energy of activation of 27.3 kcal/mol. The fast phase appeared to be linearly dependent upon NADH concentration, produced a 45 to 50% quenching of the protein fluorescence, and had an energy of activation of 19.6 kcal/mol. The results are interpreted in terms of a three-step binding reaction, corresponding to initial complexation followed by conformational changes.

Alcohol Oxidoreductases↗
PubMed1980

Resolution of the individual steps in the reaction of lysozyme with the trimer and hexamer of N-acetyl-D-glucosamine at subzero temperatures.

The reaction of hen egg white lysozyme with chitotriose and chitohexose has been investigated at temperatures to below -100 degrees C by using aqueous methanol and dimethyl sulfoxide cryosolvents. Initial investigations involving the effects of increasing cosolvent concentration on the catalytic and structural properties of the enzyme indicated that both methanol and dimethyl sulfoxide cryosolvents, at subzero temperatures, had no adverse effects on lysozyme. Time-dependent changes in the fluorescence emission of the enzyme, under nonturnover conditions, permitted the detection of two intermediates in the reaction with the trimer and three intermediates in the case of the hexamer. The fastest reaction observed for both substrates was complete within minutes at -90 degrees C and is attributed to initial substrate binding, followed by rapid isomerization to form a "loose" ES complex. The subsequent, slower reactions correspond to successive isomerizations of ES to "tighter" complexes. From dye binding displacement reactions, and comparison of dissociation constants, it is concluded that the predominant mode of binding for the hexamer at subzero temperature is productive. Rate and dissociated constants were determined for the observed reactions as a function of temperature, pH, and cosolvent concentration. Overall the reaction pathway in methanol cryosolvents at subzero temperatures appears to be very similar to that determined from investigations in aqueous solution at ambient temperatures using a variety of rapid reaction techniques. Conditions necessary to accumulate and stabilize each of the observed intermediates have been determined, thus permitting further studies to delve more deeply into the nature of the intermediates.

Acetylglucosamine↗
PubMed1979

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH.

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347--352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213--217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.

Chemical Phenomena↗
PubMed1979

Detection and accumulation of tetrahedral intermediates in elastase catalysis.

Tetrahedral intermediates in the reaction of elastase with specific di- and tripeptide p-nitroanilide substrates have been detected, accumulated, and stabilized at high pH by using subzero temperatures and fluid aqueous/organic cryosolvents. The tetrahedral adducts are characterized by spectra with lambda max of 359 +/- 2 nm, compared with thata of 380 nm for p-nitroaniline and 315-320 nm for the substrates. The maximal concentration of intermediate that could be accumulated varied with the different substrates from 40 to 100% of the active enzyme present. The pH dependence of the reactions indicated that formation of the tetrahedral intermediates was rate-limiting at low pH (pK* = 7.0 at -39 degrees C) and that collapse to the acylenzymes was rate-determining at high pH. When corrected for the effect of temperature and cosolvent, the rate of intermediate formation was in good agreement with that measured at 25 degrees C in aqueous solution by stopped-flow techniques.

Animals↗
PubMed1976

Papain-catalyzed reactions at subzero temperatures.

As a first step in the investigation of papain catalysis using subzero temperatures to detect, accumulate, and characterize enzyme-substrate intermediates, we have studied some potential cryosolvents and carried out preliminary intermediate trapping experiments. The effects of subzero temperatures and aqueous dimethyl sulfoxide solutions on the papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester have been investigated in detail. At 0 degrees C, the value of kcat decreases with increasing dimethyl sulfoxide concentration, decreasing in proportion to the decreased water concentration; however, the value of Km increases exponentially. The effect on Km can be accounted for by a combination of both dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 7.65 M (60% v/v) dimethyl sulfoxide is linear over the temperature range 0 to -45 degrees C and extrapolates to a calculated value of kcat at 25 degrees C in excellent agreement with that obtained in the absence of organic solvent. The pH-rate profile is not substantially perturbed by the presence of 7.65 M dimethyl sulfoxide. At -45 degrees C and below, turnover occurs extremely slowly, and is essentially negligible, although acylation is still quite rapid. Consequently, the acyl enzyme, Na-carbobenzoxy-L-lysyl-papain, can be readily accumulated and trapped at temperatures below -50 degrees C. At these low temperatures, under conditions of excess substrate, the amount of p-nitrophenol liberated in the acylation reaction is equivalent to the active-site normality of the enzyme, indicating a 1:1 stoichiometry in formation of the acyl enzyme. The effect of dimethyl sulfoxide up to 7.65 M, on the intrinsic ultraviolet, fluorescence, and circular dichroic properties of the enzyme shows no evidence of any solvent-induced structural changes. All experimental observations are consistent with the conclusion that 7.65 M dimethyl sulfoxide and subzero temperatures have no deleterious effects on papain-catalyzed reactions. A related series of experiments indicate that aqueous ethanol cryosolvents up to 13.7 M (80% v/v) are also suitable. Preliminary experiments at subzero temperatures using Na-carbobenzoxy-L-lysine methyl ester suggest the existence of three enzyme-substrate intermediates which can be detected and accumulated.

Circular Dichroism↗
PubMed1976

Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate.

The reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitroanilide has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvent. Following initiation of the reaction at temperatures as low as -90 degrees C, a series of four reactions prior to the normal rate-limiting step (acylation) was detected spectrophotometrically. Various experimental observations have led to the following interpretation of these reactions. Reaction 1 corresponds to the binding of substrate yielding the initial Michaelis complex. Reactions 2 and 3 are two pH-independent reactions, ascribed to substrate-induced changes in the positions of active-site groups. Reaction 4 is a pH-dependent reaction (pK = 5.9) which involves the imidazole of His-57 but which is not the formation of a tetrahedral intermediate, oxazolinone, or acyl enzyme. The slowest detected step corresponded to the acylation reaction. No evidence for the accumulation of a tetrahedral intermediate was obtained. Spectral, kinetic, and thermodynamic data for these reactions are presented, as is justification for the relevance of these findings to the reaction under physiological conditions. These results demonstrate the utility of subzero temperatures in enzyme mechanism studies, especially with regard to allowing the accumulation of intermediates which may be quite stable at appropriate values of pH and low temperature.

Anilides↗