Urease Catalysis and Structure

Abstract

Abstract Investigation of their ultracentrifugal and electrophoretic behavior and chemical reactivity has been used to characterize the suprastructure of the nongenetic urease isozymes. As purified from jack bean meal, urease is obtained in the α form (Mr = 480,000, s20,w = 18.3), which is composed of two noncovalently bound, equal and active A1 half-units (Mr = 240,000, s20,w = 11.5) which separate after treatment with glycol (and similar solvents) and are stable in neutral aqueous solution at low ionic strength. Upon removing thiol, α aggregates to an integral arithmetic, disulfide-bonded polymer series in a reaction that is fully reversed by addition of thiol. Treatment of the polymer series with 40 to 50% glycol produced an intermediate series of hemipolymers whose Mr values differed from those of the corresponding polymers by an A1 unit. Polymer and hemipolymer members have been isolated by sucrose gradient ultracentrifugation, were equiactive, and remained unchanged in form, purity, and activity for several days. Concentration dependence was not evident for any of the six isozymes for which multiple sedimentation coefficients were obtained. The hydrodynamic data were analyzed by the Kirkwood equation, Perrin's formula, and Stokes law. The analysis had indicated that the polymers were composed of a linear singlet chain of spherical α units. Taking A1 as the polymerizing unit, the Kirkwood equation best described the polymers and hemipolymers as a linear doublet chain of A1 monomers. It is concluded that the analysis is self-consistent and that urease polymerizes in a linear, disulfide-bonded condensation reaction. In addition, the evidence suggests that the isolated A1 isozyme is also nearly spherical and undergoes conformational change upon dimerizing to the spherical α-urease.