Mechanism of Molybdenum Nitrogenase.

How lipids affect the activities of integral membrane proteins

RABBIT skeletal muscle myosin is composed of two heavy chains and four light chains1. Two classes of light chains can be distinguished both chemically and functionally. Within the class of essential light chains (those which cannot be removed without loss of ATPase activity), two chemically related but pheno-typically distinct proteins have been identified2, both of which are present in single fibres of fast-twitch muscles3. The existence of both phenotypes within a single psoas muscle cell indicates that either there is a single population of myosin molecules with a different alkali light chain on each subfragment-1 head, or there are at least two populations of myosin present. Densitometric and radiochemical methods have shown that there is an unequal distribution of these two light chains; this supports the hypothesis that myosin isoenzymes occur in histochemically homogeneous muscles3–5 which cannot be attributed to the presence of contaminating slow-twitch fibres. The presence of different heavy chains also is indicated by the observation of amino acid substitutions in certain peptide sequences6,7. Thus fast-twitch muscles contain myosin showing heterogeneity of both light and heavy chains, suggesting isoenzyme populations, but so far these have not been separated, nor have the proteolytic subfragments derived from them.

Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin.

Mechanism of Blebbistatin Inhibition of Myosin II

The binding change mechanism for ATP synthase — Some probabilities and possibilities

Mounted on the Internet with the permission of the Biochemical Society (1974). Also available from http://www.biochemj.org/bj/141/0351/1410351.pdf.

/pdf/the-magnesium-ion-dependent-adenosine-triphosphatase-of-36bg3gafe5.pdf

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation.

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.

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

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate–NAD+ compound was followed by the change in E325; thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6·0 at different temperatures and ionic strengths as functions of pyruvate, NAD+ and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD+. It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme–NAD+ compound involved in the normal catalytic process.

/pdf/the-kinetics-of-the-reversible-inhibition-of-heart-lactate-ufdbbb05ks.pdf

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5.5-8.5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8.1 and 5.9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8.1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5.9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme-substrate complex.

/pdf/the-kinetics-of-the-reaction-of-nitrophenyl-phosphates-with-2e87q3gjzn.pdf

The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli.

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.

/pdf/a-substrate-induced-conformation-change-in-the-reaction-of-79irb1il8a.pdf

H. Gutfreund

Papers

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation.

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

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli.

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli