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Clinical Chemistry 21: 235-242, 1975;
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Clinical Chemistry, Vol 21, 235-242, Copyright © 1975 by the American Association for Clinical Chemistry

Evidence Supporting a Proposed Mechanism Explaining the Inverse Relationship between Guanidinoacetate and Guanidinosuccinate in Human Urine

Anthony Koller 1, Jay D. Comess 1, and Samuel Natelson 1

1 Department of Biochemistry, Michael Reese Medical Center, Chicago, Ill. 60616.

A proposed mechanism [Clin. Chem. 19, 668 (1973)] for the inverse relationship between guanidinoacetate (I) and guanidinosuccinate (II) in human urine is explored. The mechanism proposes that canavaninosuccinate (III) may be reduced to form homoserine and II or, alternatively, that the III may be acted upon by a lyase to form canavanine and fumarate. The canavanine would then proceed to transamidinate to glycine to form I. This study demontrates for the first time that lyase activity for converting III to canavanine and fumarate exists in human liver and kidney extracts. Transamidination from canavanine to glycine to form I is also readily accomplished with human tissue. Reductive cleavage of III to II and homoserine has been demonstrated before [Clin. Chem. 15, 397 (1969)]. The optimum pH for the lyase reaction is 6.5, for the reductive cleavage it is 8.7. In following the course of the lyase reaction, we developed a technique whereby the fumarate formed was hydrated with fumarase (EC 4.2.1.2) and then dehydrogenated with malate dehydrogenase (EC 1.1.1.37). The changes in absorbance of NADH formed in the reaction were then measured and used to determine the amount of fumarate formed, as a measure of lyase activity. Canavanino-succinate lyase activity follows pseudo-first-order reaction kinetics. The Michaelis constant of this lyase was 6.16 x 10-4 mol/liter, for argininosuccinate lyase 9.74 x 10-4 mol/liter. These data suggest that the binding affinity for III to the enzyme is greater than that for argininosuccinate. Glycine added to the reaction acts as an activator, probably because it removes the canavanine from the reaction mixture. On the other hand, arginine acts as an inhibitor of Ill-lyase. Other substances tested, such as canavanine, fumarate, and argininosuccinate had no effect on the reaction kinetics.


Key Words: reaction kinetics • uremia • metabolic pathways • ureidohomoserine • canavininosuccinate • canavanine • transamidination • canavaninosuccinate synthetase, reductase, and lyase • argininosuccinate lyase

Submitted on October 14, 1974
Accepted on November 20, 1974






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Copyright © 1975 by the American Association for Clinical Chemistry.