Metal-free Apoenzyme Research Articles

Metal Substitutions and Inhibition of Thermolysin: Spectra of the Cobalt Enzyme

Abstract Removal of zinc from thermolysin results in an inactive, metal-free apoenzyme. Zn2+, Co2+, and Mn2+, when added in stoichiometric amounts, restore 100, 200, and 10% of the activity of the native enzyme. Fe2+ in high molar excess restores about 60% of the native activity. Zinc is bound much more firmly than is cobalt, as shown by mutual displacement and the resultant enzymatic activities. Zinc in excess of that required to induce activity inhibits the enzyme, seemingly by binding to a specific, inhibitory site. The zinc atom does not seem to contribute to the stability of the protein toward heat denaturation. Chelating agents, e.g. 1,10-phenanthroline, inhibit native thermolysin by competing with the enzyme for its zinc atom and removing it. The inhibition is fully reversible on dilution or on addition of Zn2+ ions and is a function of concentrations of enzyme and chelating agents. The enzymatically active cobalt thermolysin exhibits absorption and circular dichroic spectra indicative of an unusual environment of the cobalt atom. There is a shoulder near 500 nm and a maximum at 555 nm (e ∼90). These data together with spectral perturbations of the CD, magnetic circular dichroic, and absorption spectra lead to the conclusion that the cobalt atom is coordinated in a distorted tetrahedral geometry. This deduction is consistent both with those other cobalt-substituted zinc enzymes which exhibit similarly unusual properties as compared with Co(II) complex ions and with interpretations of x-ray structure analysis of native thermolysin.

Metalloalkaline phosphatases from Bacillus subtilis: Physicochemical and enzymatic properties

The physicochemical, enzymatic, and metal-binding properties of the alkaline phosphatase from the gram positive organism, B. subtilis, are described. The native protein is a dimer of MW ∼100,000 containing 2.48±0.5 g atoms of zinc. The most active preparations of the homogeneous enzyme catalyze the hydrolysis of p-nitrophenylphosphate at a maximum velocity of ∼8100 μmoles/hr/mg enzyme dimer, over twice the specific activity of the crystalline enzyme from the gram negative organism, E. coli. The metal-free apoenzyme prepared by removal of the metal atom with Chelex resin is inactive. Of the first transition and IIB metals only Zn(II) and Co(II) restore activity to the metal-free apoenzyme. Addition of Co(II) results in an enzyme with twice the activity of the reconstituted. Zn(II) enzyme. The enzyme activity is stimulated threefold by increasing the ionic strength from 0.1 to 1.0. The enzyme also catalyzes phosphoryl transfer from p-nitrophenylphosphate to Tris. The visible absorption spectrum of the Co(II) enzyme shows the metal coordination site to be almost identical to that present in the enzyme from E. coli. Visible absorption maxima of the Co(II) enzyme occur at 620 (ɛ=305), 596 (ɛ=335), 567 (ɛ=500), and 517 nm (ɛ=382).

Immunologic and kinetic properties of carbonic anhydrases from various tissues

1. 1.|Rabbit antisera, specific for the highly purified forms of human (HCA B and HCA C) and bovine (BCA B) erythrocyte carbonic anhydrases (carbonate hydrolase, EC 4.2.1.1), have been produced. 2. 2.|Substances, immunologically and kinetically similar to HCA B and HCA C, were found in human blood, kidney and lens. The erythrocytes of the prenatal infant contained less HCA B than those of the adult. In the lens HCA C appeared only after solubilization, thus indicating a bimodal intracellular distribution of the isoenzymes. 3. 3.|In man, the dog and the rat, the red cells, kidney and lens exhibited a similar isoenzyme pattern. 4. 4.|The metal-free apoenzyme and the Cu and Co versions of HCA B reacted antigenically like the native enzyme, although they were kinetically different. 5. 5.|Saturating the enzyme with a sulphonamide inhibitor did not change its antigenicity. 6. 6.|The specific antiserum inhibited the catalytic activity (hydration of CO 2) of its enzyme but not wholly.