Involvement of Guanidino Groups in Anion Binding Sites and in Enzyme Catalyzed Covalent Modification Reactions

This chapter discusses the involvement of guanidino groups in anion-binding sites and in enzyme-catalyzed covalent modification reactions. It presents a study that determines whether an un-ionized guanidino group might be important for: (1) binding of phosphate, (2) its chemical reactivity, and (3) its involvement in specific enzyme-catalyzed covalent modification reactions. A guanylhydrazone was used as a reactant because it has a lower p K a value than arginine and it allows direct testing of the effect of the charge state of the reactant in chemical and enzymatic reactions. The chemical reactivity of nitrobenzylidine aminoguanidine (NBAG) with p-nitrophenylglyoxal was examined at different pH values and at a constant pH but in the presence of various detergents to determine whether the reaction occurred more readily with the un-ionized form. The reactions were followed using high-performance liquid chromatography to measure the decrease in NBAG with time. With a 20-fold excess of p-nitrophenylglyoxal, the reaction consisted of two pseudo-first-order phases. A second-order rate constant was evaluated after the contribution of the slow phase from the initial phase was subtracted. The results show that the reaction rate increases with pH and the change in rate constant nearly correlates with an increase in the amount of the unprotonated species of NBAG.