METHODS FOR DETERMINATION OF EFFECT OF 

 TEMPERATURE UPON VELOCITY CONSTANTS 

 OF ENZYME REACTIONS 



Britton Chance, Johnson Foundation, University of 

 Pennsylvania, Philadelphia, Pennsylvania 



HE BULK of the work on the effect of temperature on enzyme activity 

 has been concerned with the over-all activation energy of the catalyzed 

 reaction and with the loss of enzyme activity caused by the irreversible 

 or the reversible thermal denaturation of the enzyme. While such studies 

 are useful methodologically in determining the range over which prepara- 

 tive procedures or physical and chemical studies of the intact enzyme can 

 be made, they give little data on the nature of the actual activation 

 processes involved in the formation and break-down of the enzyme sub- 

 strate compounds involved. A summary of such data together with recom- 

 mendations on the necessity for measuring the effect of temperature upon 

 particular velocity constants involved in the formation and break-down 

 of the enzyme substrate compound was made by Sizer in 1943 (1). The 

 significance of the reversible denaturation in various enzyme-catalyzed 

 reactions or processes, as influenced by heat, pressure and chemical agents, 

 has been discussed by Johnson, Eyring and Polissar (2). 



A better understanding of the role of intermediate compounds in en- 

 zyme action, together with the development of more sensitive methods 

 for studying enzyme reactions gives, in a few cases, critical data on the 

 effect of temperature on individual reaction velocity constants. Only when 

 the chemical reaction sequence has been specified in some detail can any 

 rigorous, detailed use be made of such thermal data. Thus, this con- 

 tribution stresses primarily the development of methods suitable for 

 measuring temperature effects on individual velocity constants in enzyme 

 action and gives as examples some of the experimental data that have 

 been obtained. 



The classic method for the determination of the effect of temperature 

 upon enzyme reactions has been based upon a study of the overall reac- 

 tion in which the disappearance of substrate or the formation of product 

 is measured. In only a few enzyme reactions, however, can the overall 

 velocity be attributed to a particular step in the reaction over reasonable 

 ranges of substrate concentrations. Moreover, when temperature is varied 

 over a wide range, as is necessary for accurate determination of reaction 

 heats, the rate determining step may shift from one point in the reaction 

 sequence to another. 



37 



