THE OXIDATION MECHANISM OF GLYCOLIC AND l-LACTIC ACIDS BY THE ENZYME FROM LEAVES

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1. The amounts of O₂ consumed and CO₂ evolved in the oxidation of glycolic acid by the salt-precipitated enzyme were found to be dependent upon the amount of catalase contaminated in the enzyme sample. When catalase was practically absent, the amount of O₂ consumed per mole of glycolic acid oxidized was one mole. When excess of catalase was present the O₂-uptake was reduced to 1/2 of this value.
2. The addition of cyanide caused the accumulation of H₂O₂ together with the disappearance of CO₂-formation without changing the amount of O₂-uptake. The addition of azide, on the other hand, caused the increase in CO₂-formation with no H₂O₂-accumulation, but the same O₂-uptake was brought about as in the case of CN-addition.
3. The addition of peroxidase and its hydrogen donor (reduced Bindschedler's green) caused the disappearance of CO₂-formation and of H₂O₂-accumulation without changing the amount of O₂-uptake.
4. H₂O₂ was proved to react spontaneously with the intermediate as well as with synthetic glyoxylic acid to form CO₂. Some additional evidence was aduced in support of the conclusion that glyoxylic acid is the intermediary product in the oxidation of glycolic acid.
5. Glycolic acid is oxidized by plant sap with an equivalent amount of O₂-uptake but only with a little CO, -formation. When the sap was pretreated with H₂O₂, CO₂-formation was remarkably increased.
6. The formation of H₂O₂ and accumulation of pyruvic acid were confirmed in the oxidation of lactic acid by the same enzyme in the presence of catalase in excess.
7. Based on these observations it was concluded that the oxidation of glycolic and lactic acids proceeds as follows:
In the enzyme solution,
Glycolic acid+O₂→_oxidaseglyoxylic acid+H₂O₂→_spontaneousformic acid+CO₂+H₂O,
Lactic acid+O₂→_oxidasepyruvic acid-+H₂O₂→_spontaneousacetic acid+CO₂+H₂O.
In plant sap,
Glycolic acid+O₂+AH₂^oxidase→_peroxidaseglyoxylic acid+A+2H₂O,
Lactic acid+O₂+AH₂^oxidase→_peroxidasepyruvic+A+2H₂O,
where AH₂ represents some unknown, presumably phenolic, substances present in the plant sap.
The authors wish to thank Prof. Dr. Hiroshi Tamiya for his kind advice and interest in this work. The authors are also indebted to Dr. Yasuyuki Ogura for furnishing purified equine liver catalase used in this study, and to Dr. Takeshi Mori for his helpful suggestions in the purification of the enzyme.
This work was aided by a Grant from the Scientific Research Fund of the Ministry of Education.