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Molecular Pharmacology, Vol 10, 648-656, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Human Erythrocytic Hypoxanthine-Guanine Phosphoribosyltransferase: Effect of pH on the Enzymatic Reaction

CHONG M. KONG 1 and R. E. PARKS JR. 1

1 Section of Biochemical Pharmacology, Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912

The effect of pH on human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was studied, and the enzyme was found to be unstable at pH values below 5.5 and above 9.0. The enzyme exhibited marked differences in pH optima when the various purines or purine analogues were used as substrates: 8-azaguanine, 6.5; 6-mercaptopurine, 7.5-8.0; 6-selenoguanine, 7.5-8.0; 6-thioguanine, 8.0-9.0; guanine, 8.5-9.0. The enzyme was subjected to a Dixon analysis, in which the Michaelis constants and maximal velocities were determined for these substrates at various pH values between 5.5 and 9.5. Plots of the negative logarithm of the Michaelis constants (pKm) against pH resulted in a family of graphs with downward bends. With the exception of guanine, the bending points of the curves occurred at pH values which closely approximated the pKa values of the respective compounds. Tht Vmax values increased with increasing pH within the range examined. This effect of pH on Km values suggests that the ionization state of purines or their analogues is an important factor for binding of the substrate to the enzyme. It also indicates that the undissociated form of the molecule is the effective substrate for the enzymatic reaction. When Km values are expressed in terms of the concentration of the un-ionized form, the pKm, plots result in straight lines in all cases.

Note:
ACKNOWLEDGMENTS The authors wish to thank Dr. Sungman Cha, Dr. R. P. Agarwal, and Dr. Gerald Crabtree for their helpful criticisms and suggestions, Dr. S.-H. Chu for his gift of 6-selenoguanine, and the Division of Hematological Research, Pawtucket Memorial Hospital, for supplying fresh human erythrocytes.

Submitted on March 28, 1974






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