Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.

Bovine erythrocyte superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu2+ and Zn2+ in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu2E2SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zincfree protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu2Cu2SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30°C are also consistent with formation of Cu(II)Im-Cu(II). A plot of changes in the signal amplitude of g1 for Cu2E2SODase as a function of pH gives an apparent pKa of 8.2 for the transition. The long-wavelength absorption with Xmax = 700 nm characteristic of Cu2E2SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu2-Cu2SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO3, although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu2+ from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu2Zn2SODase, and the four-copper protein, Cu2-Cu2SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu2E2SODase and its thermal stability are reexamined in light of these new findings.

Nuclear magnetic resonance (NMR) spectroscopy of the exchangeable protons, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O has been found to be a powerful method to study the active site of the enzyme. This technique has been employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that zinc alone organizes the active site structure. All eight histidines per subunit of apoenzyme react with DEP. The accessibility of these residues to solvent is borne out by the broad, featureless NMR spectrum of the apoprotein. In the holoenzyme only His-19, which is exposed to solvent, can be modified with DEP. The reduced holoenzyme shows a well-resolved NMR spectrum compared with the oxidized form in which the lines are broadened by the paramagnetic copper ion. A spectrum very similar to that of the reduced enzyme is generated by addition of one zinc ion per subunit of apoprotein showing that zinc alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of zinc per subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios greater than or equal to 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.

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