Purification and Characterization of Component S of Glutamate Mutase

Abstract

Abstract Glutamate mutase from Clostridium tetanomorphum contains two different protein components or subunits. Purification of Component E has been previously reported. This paper describes the purification of the second protein, Component S, approximately 350-fold in 20 to 25% yield. The purified protein shows no detectable β-methylaspartase or Component E activity, and contains no significant amount of vitamin B6 or vitamin B12 derivatives. When in the reduced state, the enzyme appears to be at least 90% pure. The molecular weight of Component S was found to be 17,000 ± 1,000 by sedimentation equilibrium, sedimentation velocity, and gel filtration techniques. The amino acid composition of the protein was determined. Component S requires incubation with thiols for maximal activity and is inhibited by iodoacetate and arsenite. Out of a total of 5 sulfhydryl groups in the native protein, 1 or 2 appear to be essential for activity. These sulfhydryl groups readily undergo reversible oxidation to form dimer molecules and other inactive oxidized forms when the protein is exposed to air in the absence of thiols. However, the protein is most stable when stored in the absence of thiols. The effect on mutase activity of adding increasing amounts of either component to a fixed amount of the other component suggests that an equilibrium exists between the two components and the active complex. No inhibition occurs at superoptimal levels of either component when they are sufficiently purified. Half-maximal activity is obtained when the molar ratio of Component E to Component S is approximately 1. The insensitivity of Component E to iodoacetate inhibition suggests that the requirement of the mutase reaction for thiols reflects only the necessity of maintaining the sulfhydryl groups of Component S in the reduced state. Greater activity is obtained when Component S is incubated with mercaptoethanol before addition of other assay components than with any other order of addition of the thiol. Increasing the S:E molar ratio 50-fold, from about 0.3 to 16, was shown to decrease the Michaelis constant for the benzimidazolylcobamide coenzyme 40-fold. Binding experiments with dimethylbenzimidazolylcobamide coenzyme showed that Component E alone binds coenzyme weakly, but much more coenzyme is bound when an excess of Component S is present. Component S alone binds little, if any, coenzyme. No stable complex between the mutase components could be detected by gel filtration techniques. No spectral changes were observed upon addition of coenzyme or coenzyme plus substrate to the enzyme components, either separately or together.