Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

Abstract

The reactivity and the mode of activation of the essential – SH group (Cys‐149) of d‐glyceral‐dehyde‐3‐phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187–190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys‐149 of NAD‐free d‐glyceraldehyde‐3‐phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys‐149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK₁= 5.5, pK₂= 8.2). It is concluded that there are two reactive forms of the –SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion‐pair composed of a mercaptide ion and some base, possibly the imidazolium group of His‐176. The ion‐pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK₂= 8.2. The ion‐pair decomposes to a nondissociated thiol group and a protonated base with pK₁= 5.5. In the presence of NAD, only the pH‐rate profile of alkylation of d‐glyceraldehyde‐3‐phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK₁ and an upward shift in pK₂ indicate that the ion‐pair exists in a somewhat wider pH‐range in the enzyme‐coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.