β-D-Galactosidase (E.C.2.2.1.23) of $\underline{E}$. $\underline{coli}$ strain B was partially purified and characterized for the further study of the enzymic reaction mechanism. The increase of the specific activity by the subsequent purification thru the granulated hydroxy-apatite column chromatography was 15 folds of its crude fraction and it was shown that the general characteristics were in accord with the results of the other reports: catalytic specificity on some β-D-galactosides, and 6.8-7.2 as optimum pH, 40℃ as the optimum temperature, 1.01 - 1.14mM for Km, inhibitory effects by the chelate agents such as ethylenediamine tetracetate and citrate and by $\underline{para}$-chloromercuribenzoate and iodoacetamide known as the compounds modifying sulfhydryl groups.
For the purpose of verifying the role of carboxylate group supposed to be in the active site, the results, of the treatment by hydroxylamine, the chemical reagent modifying carboxylate group in protein, demonstrated non-competitive inhibition caused by the irreversible modification of carboxylate group, the reduction of hydryxylamine-inhibitory effect by magnesium (II) ion seemed to be protective against the hydroxylamine action to the carboxylate group non-significant deviation of the hydrolytic activity of $\mbox{\underline{para}}$-nitrophenol- β -D-glucoside ($C_4$ -epimer of the correspondent substrate)by the treatment in comparing before the treatment, and the non-competitivity in the inhibition by para-nitrophenol-β-D-glucoside for the treated-enzymatic catalysis of orthonitrophenol-β-D-glucoside. By the above results with the uncompetitive inhibition by cellobiose for this native enzyme, it is postulated that the carboxylate group in the active site be involved in the binding with $C_4$-hydroxyl group of glycon of this enzyme substrate-β-D-galactoside.
In conclusion, the above results of the indirect and binding-characteristical involvement of carboxylate group can be a powerful support for the model of mechanism of this enzyme catalysis to be $\mbox{\underline{via}}$ epoxide intermediate with C2-hydroxyl group participation, to be specific for C1-β-anomeric glycoside having correspondences with the imidazole and the sulfhydryl groups in the catalytic site with those above accordances of this enzyme properties in comparison to other $\mbox{\underline{E}}$ $\mbox{\underline{coli}}$ enzymes, and to be proper to the mechanismic models containing other substep procedures reported previously.
