MODIFICATION OF CARBONIC-ANHYDRASE-III ACTIVITY BY PHOSPHATE AND PHOSPHORYLATED METABOLITES

Chicken carbonic anhydrase III (CA III) is potently activated by phosp hate (k(cat)/K-M is increased for bicarbonate dehydration from 0.6 x 1 0(4) M(-1)s(-1) to 1.2 X 10(4) M(-1)s(-1) in 10 mM phosphate). The pre sence of phosphate both reduces K-M and increases k(cat). Activation i s also evident when the enzyme is pretreated by incubating with phosph ate and is subsequently assayed at negligibly low phosphate concentrat ion. The dissociation of the phosphate-enzyme complex is slow (k(d) = 0.084 min(-1)) suggesting that activation in vivo may be sustained thr ough fluctuating phosphate levels. A similar enhancement may also be a chieved by various phosphorylated metabolites at concentrations within homeostatic physiological levels. Modification of active site arginin e residues by 2,3-butanedione (MD) yielded similar results to those ob tained by pretreatment with phosphate, suggesting a common feature of the activating mechanism. Both DD-modified and phosphate-modified enzy me could be further activated by augmenting the assay mixture with pho sphate. Two mechanisms of phosphate enzyme interaction are inferred. P hosphate has no significant effect on the esterase activity, thus supp orting the premise that the esterase and CO2 hydration sites are physi cally separated in the CA III isoenzymes.