Year: 1953
Type: article
Abstract: A study ofthe variation with temperature and pH of such properties of an enzyme as its maximum velocity and affinities for substrates and inhibitors might be expected to throw valuable light on the mechanism ofthe reaction catalysed by the enzyme.In this paper are reported some results on the variation of maximum velocity and Michaelis constants with temperature of crystalline fumarase, as well as the variation of Michaelis constants with pH.The variation of activity with pH in the presence of various salts has already been reported (Massey, 1952), and the variation of inhibitory constants of a number of dicarboxylic acids with both temperature and pH will be reported in a future paper.METHODS Fumarase.The fumarase solutions used were of twice recrystallized material obtained from pigs' hearts as already described (Massey, 1952).E8timation of enzyme activity.Details are given in the second paper of this series (Massey, 1953).All the values reported here are initial velocities, when the reaction rate is still constant.Activating anion8.All the experiments reported here, unless stated to the contrary, were carried out in the presence of 0-067 M-phosphate buffers.Temperature control.The temperature was controlled to within +0-10 of the desired temperature by means of an apparatus designed by Dr M. Dixon, F.R.S., which will be described in a separate communication. Determination of the ionization con8tant8 of fumaric acid.These were determined by electrometric titration of 0-02Mfumaric acid with C00-free 0-025w-NaOH.The temperature was maintained constant to within ±0-10 by immersion in a thermostated water bath.Stirring of the reaction mixture was accomplished by a constant stream of C02-free N2, to prevent the absorption of C02 from the atmosphere into the reaction mixture.The titration was followed by measuring the pH with a 'Cambridge' electrometer, the power for which was supplied by a 12 V. storage battery.The instru- ment was standardized at the particular temperature investigated with 0-05M-potassium hydrogen.phthalate
Source: Biochemical Journal
DOI: 10.1042/bj0530072
Author: V. Massey
Institution: University of Cambridge
Language: English
Topic: Chemical Reaction Mechanisms
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W95444506