Essential Role of Thiol Groups in Carboxylase

Abstract

ESSENTIAL thiol groups of enzymes are usually detected by the inactivation of the enzyme with sulphydryl reagents, such as substances forming mercaptide, or oxidizing or alkylating substances¹. Yeast carboxylase seems to be affected in a rather uneven and contradictory way by sulphydryl group detectors since 4-aminophenyldichloroarsine², p-chloromercuribenzoate³, iodine³, iodoacetate³, cupric^3,4, mercuric and silver ions⁴, or quinone⁵ inhibit the enzyme, while in other experiments, lewisite oxide⁶, ethylarsenoxide⁷, iodoacetate⁴ or ethyl iodoacetate⁸ do not modify its activity significantly. It must be pointed out: (a) that inhibition by a single thiol reagent does not prove the existence of sulphydryls, since even very selective mercurial compounds⁹ can inactivate enzymes which by other evidence do not seem to contain essential thiol groups; and (b) that the effect of sulphydryl detectors often depends on the purity of the enzyme preparation tested¹⁰, a circumstance neglected in some of the work quoted. Therefore, considering the essential part played by thiol groups in most of the enzymes related to pyruvate metabolism², it seemed interesting to make a systematic study of the action of several types of thiol reagents on a conveniently purified carboxylase, and for that purpose we have used the preparation described by Green et al.⁴ ($Q_{\text{C}{\text{O}}_{\text{2}}}$ about 12,000).