|Baker, J.J.; van der Drift, C.; Stadtman, T.C.
|Purification and properties of beta-lysine mutase, a pyridoxal phosphate and B12 coenzyme dependent enzyme
|The cobamide coenzyme dependent beta-lysine mutase has been purified to homogeneity by fractionation with ammonium sulfate and chromatography on DEAE-cellulose, Sephadex G-150, and DEAE-Sephadex A-50. The native enzyme is a tetramer (mol wt 170,000) composed of two sub- units of approximately 32,000 and two of approximately 52,000 daltons. The enzyme exhibits an absolute dependency on pyridoxal phosphate for activity. During isolation there is degradation of enzyme-bound cobamide coenzyme to hydroxy(adeny1)cobamide which is a strong inhibitor and remains tightly bound to the protein. Incubation with co- balamin coenzyme, Mg2+, a mercaptan, and pyridoxal phos- phate displaces the hydroxycobamide and markedly activates the mutase. A monovalent cation (e.g., K+) is required for catalytic activity of the mutase. Low concentrations of hy- droxylamine and other carbonyl reagents inhibit the mutase. presumably by reacting with pyridoxal phosphate. During catalysis under conditions where the mutase product, 33- diarninohexanoate, is not continuously removed there is extensive cleavage of cobalamin coenzyme to free 5 ’-deoxy- adenosine and a cobalamin with concomitant inactivation of the enzyme. This inactivation is prevented by the addition of a sulfhydryl protein (E2) and ATP. Inactivation of the mutase during catalysis is greatly accelerated by oxygen.