Carbon isotope effects in the glycine decarboxylase reaction in vitro on mitochondria from pea and spinach

Two independent experiments were made to:study-carbon isotope effects (C-13 /C-12) in the reaction of enzymatic decarboxylation of glycine in vitro on mitochondria, isolated from pea (Pisum sativum L,) and spinach (Spinacia ol eracea L.) leaves. Experiments were run at 25 and 37 degrees C at pH graded from 6.8 to 8.0 with a 0.2 interval. Various combinations of cofactors (py ridoxal 5-phosphate, ADP, and NAD(+)) were used. The difference in carbon i sotope between CO2 and the carboxyl: group of glycine varied from 11.8 to - 8.5 parts per thousand. Upon variations in pH and cofactors, the carbon iso tope composition of the CO2 produced during glycine decarboxylation showed similar changes in mitochondria from different plant: species. Shifts in th e carbon isotope composition of CO2 were always positive in the aforementio ned pH range and reached a maximum at pH 7.0-7.4. NAD(+) promoted the great est isotope effect of all the cofactors tested. The removal of NAD(+) from the reaction medium significantly enriched evolved CO2 with C-12 isotope an d could even invert the sign of the effect, which indicates that disruption of the C-C bond became the rate-limiting stage in this reaction. The isoto pe effects in the reaction of enzymatic decarboxylation of glycine are:disc ussed on the basis of the supposed mechanism of the reaction. The results a re consistent with the possible-occurrence of negative isotope effects in t he reaction of glycine decarboxylation bt vivo and their relation to photor espiration.