Purification and Properties of Component E of Glutamate Mutase

From the ¹ From the Department of Biochemistry, University of California, Berkeley, California

A method is described for the purification of Component E of glutamate mutase about 180-fold with an 18% yield. The purified component shows no ß-methylaspartase activity and is virtually free of the gel supernatant (S) component of the mutase. The most highly purified preparations contain about 75% of one protein and 25% of a second protein as judged by ionophoresis and ultracentrifugation. The activity is associated with the major component. Sedimentation analysis of the major component gave an s_20,w value of 6.90 and a calculated molecular weight of approximately 128,000. Component E preparations are light pink, apparently because of contamination by corrinoid vitamins. They do not contain significant amounts of pyridoxine or related B₆ vitamins. Component E is rather stable to storage under several conditions but is rapidly inactivated by freezing in the presence of mercaptoethanol or by incubation at 0° with some cationic buffers, such as tris(hydroxymethyl)aminomethane and imidazole. Inactivation by Tris is largely prevented by 50 mm l-glutamate, methylaspartate, or, less effectively, by l-aspartate; neutral amino acids appear to be ineffective. The results indicate that glutamate and methylaspartate have a special affinity for the E component.

Both purified Component E and partially purified Component S are essential for glutamate mutase activity under either aerobic or anaerobic assay conditions. For maximal activity, at least one of the components must be reduced by incubation with mercaptoethanol or another sulfhydryl compound before it is used in the assay; reduction of either component is about equally effective. The addition of 1 to 3 mm mercaptoethanol to the spectrophotometric assay solution is also beneficial. The influence of the relative amounts of the E and S components on mutase activity has been determined. There is an optimal amount of Component S which increases with the amount of Component E in the assay system. Superoptimal amounts of Component S inhibit markedly. The interaction of Component S with Component E to cause activation or inhibition is increased by higher levels of mercaptoethanol. The binding of B₁₂ coenzyme to Component E was investigated by two methods; both indicate that the binding is relatively weak.

The possibility that free ammonium ion or -ketoglutarate is an intermediate in the glutamate mutase reaction was eliminated by tracer experiments showing that neither ¹⁵NH₄⁺ nor -ketoglutarate-¹⁴C is incorporated into the products.

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