The effect of carbonic anhydrase on the action of yeast carboxylase

Abstract

The derivation is given of equations from which the full theoretical effect of carbonic anhydrase on the increase of manometric pressure during decarboxylation may be calculated. It is assumed that the decarboxylation, as of strong pyruvate, is a linear function of the time over the observation period. Such equations may be further developed for special conditions where the decarboxylation is a definite, but non-linear function of the time. A carbonic anhydrase prepn. was introduced from the manometer side arm, being present there in fluid having otherwise the same composition as the central fluid. This contained carboxylase prepared according to the procedure of Green et al., acting on 0.67 [image] pyruvate at pH 7.0 and 18[degree]. The production of pressure was nearly linear over the time of observation. With the introduction of active carbonic anhydrase there was a rapid rise of pressure which after a few min. proceeded practically parallel with the previous curve, or with that using inhibited carbonic anhydrase. The mean increase of pressure level, after carbonic anhydrase action, approaches but does not quite reach the expected figure when carbonic acid (or HCO3) is the primary product of decarboxylation. Using a crude water extract of dried yeast as the carboxylase prepn. and the "yellow fluid" as that of carbonic anhydrase, there was a fall of pressure on introducing the latter. This is in qualitative agreement with the experiments of Krebs and Roughton (ibid. 43:550. 1948) and shows a primary production of CO2. The degree of fall was only a fraction of that to be expected if CO2 was the sole primary product of the CO2 system. Expts. were also carried out with strong concns. of alpha-ketoglutarate and oxaloacetate used as carboxylase substrates. With the introduction of the carbonic anhydrase prepn. there occurred a small fall of pressure indicating CO2 as mainly the primary product, but the curves of pressure were not regular. Using diluted blood as the source of the carbonic anhydrase, the result with oxaloacetate was similar to the theoretical picture when CO2 is the primary product of decarboxylation. Similar results with alpha-ketoglutarate indicated a primary production mainly of CO2, but to a lesser degree than with oxaloacetate. The conclusion may be drawn that in the decarboxylation of keto-acids by yeast carboxylase, CO2 and carbonic acid (or HCO3) are both primary products, but one or the other may predominate depending on the total conditions.