1. The effect of methanol on the β-galactosidase-catalysed hydrolysis of some nitrophenyl β-d-galactopyranosides has been studied under steady-state conditions. 2. The initial fractional rate of increase of kcat. as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl β-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in kcat. at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pKa approx. 9.

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