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H Gutfreund

Publications and source records attributed to H Gutfreund.

At least 37 records · Page 2Linked to original sources
PubMed1984

Kinetics of acto-S1 interaction as a guide to a model for the crossbridge cycle.

Recent experiments on the kinetics of the interaction between myosin subfragment 1 (S1) and F-actin in solution are summarized. It is concluded that, at every step of the ATPase cycle, the association between the two proteins takes place in two stages. The equilibrium constant of the second step and thus the affinity of S1 for actin changes from step to step during the enzymatic reaction. It is proposed that the transient kinetic evidence can be interpreted in terms of two different classes of contraction models. The first one, which is widely used at present, identifies particular steps in the enzymatic reaction as directly responsible for the conformational change which represents the power stroke of muscle contraction (direct coupling model). In the second class of model, to which we wish to draw attention, changes in affinity modulated by the enzymatic reaction result in changes in the relative amounts of time spent by parts of the myosin molecule in two different environments. These environments determine whether the molecule exists in the 'long' or 'short' state, and it is the transition between these two which constitutes the power stroke (indirect coupling model).

Actins↗
PubMed1984

Kinetic studies on the formation and decay of metarhodopsins from bovine retinas.

The kinetics of the equilibration of MRI in equilibrium MRII were studied over a range of temperature and pH. Two methods were used to evaluate the relaxation time of this process. The rate of disappearance of MRI or the rate of appearance of MRII were measured after a light flash. The established equilibrium between these two intermediates at low temperatures was subjected to pressure perturbations. This latter technique was used to show that the equilibrium was truly reversible and to determine the relaxation time. From these experiments the equilibrium constants and forward and reverse rate constants were evaluated under different conditions. The simplest interpretation of our results is that the reaction can be described by a single kinetic step. This step involves a conformation change which is accompanied by a change in pK of approx. 3 units of an ionizing group of rhodopsin, resulting in the uptake of a proton during the formation of MRII.

Animals↗
PubMed1980

G.S. Adair.

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Hemoglobins↗
PubMed1978

Pressure relaxation of the equilibrium of the pig heart lactate dehydrogenase system.

The relaxation behaviour of a system of reactants equilibrated in the presence of pig heart lactate dehydrogenase was studied after pressure perturbation. Two relaxations were observed when protein fluorescence was recorded, but only the slower relaxation was apparent in observations of A340. The faster relaxation therefore involves transfer between free and enzyme-bound NADH, whereas the slower relaxation represents the reduction of NAD+. Both relaxations were observed in Tris buffer, where there is little effect of pressure on pH, and in phosphate buffer, where pH changes are significant; however, the amplitudes depended on the buffer used. The slower reciprocal relaxation time increases with increasing total enzyme concentration and decreases slightly with increasing NAD+ concentration. Computer simulations, based on a proposed mechanism, were compared with the experimentally determined amplitudes and relaxation times as a test of the mechanism.

Animals↗
PubMed1977

The consequences of nucleotide binding to liver alcohol dehydrogenase.

Extensive and informative steady-state kinetic investigations of the mechanisms of horse liver alcohol dehydrogenase have recently been complemented by observations of the fluorescence and spectroscopic characteristics of transient intermediates by rapid-reaction techniques. In this way it was possible to study separately steps involved during enzyme-substrate complex formation and during the catalytic process. It can be shown that a proton is liberated during complex formation before the transfer of a hydride ion from ethanol to form NADH. This must be due to a change in pK of a group on the enzyme protein and is linked to a change in tryptophan fluorescence. Pressure relaxation techniques have enabled us to study the rate constants of the change in tryptophan fluorescence linked to NAD+ binding and proton dissociation. We have shown that NAD+ binding occurs in two steps: a rapid secondorder association, followed by the substrate-induced isomerization to form the reactive enzyme-substrate intermediate. The isomerization rate constants were determined in both directions and their role in the overall reaction mechanism could be identified.

Alcohol Oxidoreductases↗
PubMed1976

31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.

31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.

Alkaline Phosphatase↗
PubMed1975

Quenching of protein fluorescence by transient intermediates in the liver alcohol dehydrogenase reaction.

The addition of saturating concentrations of NAD-+ and alcohol to liver alcohol dehydrogenase in a stopped flow fluorimeter results in a triphasic quenching of enzyme fluorescence. A rapid quenching occurs with a rate constant of 300 to 500 s-minus 1, followed by a slower reaction at 50 to 100 s-minus 1, and ultimately followed by a very slow reaction. The addition of NAD-+ to enzyme in the absence of substrate causes a rapid quenching of enzyme fluorescence at 300 to 500 s-minus 1, with the same amplitude as the rapid phase in the presence of substrate. These studies demonstrate that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that proton release is probably coupled to the conformational change.

Alcohol Oxidoreductases↗
PubMed1975

Energy changes during the formation and interconversion of enzyme-substrate complexes.

The rate constants and equilibrium constants of the individual steps of several enzyme reactions may be determined by the application of rapid reaction methods and isotope techniques. This makes it possible to complement the formalism of the Haldane relation with details of the reaction mechanism. It has been shown that, in several enzyme reactions, steps involving chemical catalysis are fast and have small free-energy changes compared with those of the substrate binding and product dissociation processes. Data are presented in this paper for three enzyme reactions for which different methods have been used to elucidate the kinetic parameters of the elementary steps. For cardiac lactate dehydrogenase (EC 1.1.1.27), absorption and fluorescence spectroscopy have been used to distinguish the step involved in the chemical process from those involved in the formation of the substrate complex and the release of the product. The rate of interconversion between enzyme-bound substrates and products is fast compared with other steps and the equilibrium constant for the process is near unity. Consequently, the difference of standard free energy changes for the formation of the two ternary complexes correspons approximately to the overall free-energy change of the hydrogen transfer reaction. Isotope kinetic techniques can be used to study the reactions of triosephosphate isomerase (EC 5.3.1.1). With this enzyme, the interconversion of enzyme-bound substrate into product is comparable in rate to product dissociation. The reactions of myosin subfragment 1 with ATP, studied by fluorescence spectroscopy and chemical quenching, follow a similar pattern in that the equilibrium constant of the chemical step in which water reacts with protein-bound ATP is 9. In this case, however, there is a remarkably large free-energy change associated with a first-order process involved in the binding of ATP. The possible significance of these results to energy transduction in muscle contraction as well as in the biosynthesis of ATP is discussed.

Adenosine Triphosphatases↗