Article

A Chemical Relaxation Study of Human Prostatic Acid Phosphatase

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Abstract

Chemical relaxation methods and a dilution technique were applied to the study of the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase. Although the reaction mechanism was not elucidated, rate constants and equilibrium constants were obtained for the reaction of enzyme and p-nitrophenol to form a complex. A slow, 2-sec relaxation effect which showed no concentration dependence was observed in various reaction mixtures, including some lacking the substrate and products of the hydrolytic reaction. The conclusion drawn is that there are two forms of the prostatic enzyme, which are normally in equilibrium with each other, but which undergo a relatively slow interconversion when this equilibrium is perturbed. A preliminary calculation indicates that these forms are present in the equilibrium ratio of 2:1.

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Article
A method has been devised for measuring the initial rate of hydrolysis of adenonsine monophosphates by acid phosphatase. This utilizes the enzymatic deamination of adenosine by exogenous adenonsine deaminase and permits the fall in absorbance at 265 nm to be continuously monitored.The reaction is extremely sensitive and activity was proportional to the amount of enzyme taken in the case of human prostatic and potato acid phosphatases, but not in the case of rat liver and human kidney preparations.The velocities with different monophosphates of adenosine were studied for the above enzymes and for calf intestinal alkaline phosphate. The latter showed no preference with respect to position of the phosphate ester, but the prostate, kidney, and potato enzymes showed a preference for 3′AMP while the liver enzyme hydrolyzed 3′- and 5′AMP at similar rates.Analysis of kinetic constants suggested that the velocities were more dependent upon rate of dissociation of enzyme-substrate complex than changes in the substrate-enzyme affinity.Preliminary studies suggested activation by adenine and inhibition by guanine these effects possibly being mediated allosterically at a site other than the catalytic center.Glycerophosphates inhibited almost completely AMP hydrolysis by potato and prostatic enzymes, but liver retained considerable activity towards 3′-and 5′AMP.
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Article
The analysis of multi-step reaction kinetics—an important problem in the study of biological systems—is inherently complex and requires the techniques of matrix algebra. The recent development of chemical relaxation methods allows the experimental investigation of such systems, even when some or all of the individual steps are extremely rapid. A general treatment of the theory of chemical relaxation in multi-step systems of any order and any degree of complexity is presented in this paper. The theory is developed in terms of the groups of chemical constituents that participate in the elementary reactions. A general derivation is given for the form of [KL], the matrix which determines the kinetic behavior of the system near the stationary state. A proof is given that the eigenvalues of [KL], the negative reciprocals of the relaxation times, are all either negative or zero if detailed balance (microscopic reversibility) holds at the stationary state. The number of zero eigenvalues is shown to be determined by constraints related to conservation of mass for the system and by linear dependences among the groups of reactants. Diagonalization of [KL] expresses the behavior of the system in terms of normal coordinates, each with its own relaxation time. Apparent oscillations, due to the superposition of relaxation curves for the normal coordinates, are discussed.
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