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Biomedical subjects

H Gutfreund

Publications and source records attributed to H Gutfreund.

At least 73 records · Page 4Linked to original sources
PubMed1968

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

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.

Alkaline Phosphatase↗
PubMed1968

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme-oxidized nicotinamide-adenine dinucleotide-pyruvate compounds.

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 E(325); 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.

Chemical Phenomena↗
PubMed1968

The resolution of some steps of the reactions of lactate dehydrogenase with its substrates.

1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD(+) and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD(+) per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme-NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided.

Animals↗
PubMed1966

Optical and chemical identification of kinetic steps in trypsin- and chymotrypsin-catalysed reactions.

Previous interpretations of the mechanism of trypsin- and chymotrypsin-catalysed reactions in terms of two intermediates, the Michaelis complex and an acyl-enzyme, were based on steady-state studies and on the observation of individual steps under sub-optimum conditions. In the present paper new methods for the rapid analysis of chemical events and for the spectrophotometric detection of individual steps are applied to these two enzymes. These methods can be used to study reactions with specific amino acid ester substrates. It can be shown that there are at least three distinct steps between the Michaelis complex and the release of ethanol; the latter is likely to correspond to acyl-enzyme formation. The relative rates of these three steps are measured by rapid-flow techniques from observations of the displacement of chromophoric inhibitors and reactions with specific substrates containing chromophores, as well as from ethanol analyses during a single turnover of the enzyme reactions. It is concluded that the reactions of trypsin and chymotrypsin with their specific substrates involve the formation of a specially reactive conformation of the enzyme-substrate complex and that the rate constants involved in this rearrangement are at least as important for the overall reaction as those of the subsequent formation and decomposition of the acyl-enzyme.

Arginine↗
PubMed1966

The catalytic-centre activity and kinetic properties of bovine milk alkaline phosphatase.

1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.

Alkaline Phosphatase↗