EFFECTS OF TEMPERATURE: ENZYMES 753 



to groups at the active center but to groups over the entire enzyme since 

 the net charge will be temperature-dependent. The electric field at the 

 active center that results from all the charged groups on the enzyme will 

 thus change with temperature and this will affect the interactions with in- 

 hibitors. 



(E) Changes in the configuration of the active center. The internal structures 

 of most simple substances undergoing reactions do not change appreciably 

 with temperature in the physiological range. However, proteins are not 

 rigid molecules and it is likely that shape changes occur during variation 

 of the temperature, and not only changes of the over-all shape but changes 

 in the configuration of the active centers of enzymes. More and more evi- 

 dence for enzyme flexibility is accumulating. Koshland (1959) has presented 

 some very interesting reasons for believing that certain active centers may 

 be sufficiently flexible to adjust their structures to the substrate that is 

 bound. Such self-induced fits would apply to inhibitors as well. If active 

 centers are amenable to structural changes, it is reasonable that changes 

 in temperature could bring about modifications at these regions, or, per- 

 haps, alter the inherent flexibility of the enzyme so that induction of fit 

 by inhibitors could be more or less easy. Another possibility suggested by 

 Sizer (1943) is that some enzymes may exist in two or more forms of dif- 

 fering activities, the equilibrium between these forms being temperature- 

 dependent. These different forms would presumably be susceptible to in- 

 hibitors to varying degrees. 



Examples of the Effects of Temperature on Enzyme Inhibition 



Before discussing the results obtained with enzymes it may be interesting 

 to mention some of the temperature studies on the binding of small mole- 

 cules and ions to proteins. In general it has been found that such binding 

 is not markedly affected by changes in the temperature. The binding of 

 the first copper ion to serum albumin is quite tight {AF = — 5.91 kcal/ 

 mole) but this is due to a large gain in entropy (JS = 29.2 cal/mole/degree) 

 so that JH is only 2.78 kcal/mole (Klotz and Curme, 1948). Thus more Cu++ 

 was found to be bound at 25° than at 0° but the change in binding was 

 not very large. The binding of methyl orange and azosulfathiazole to serum 

 albumin showed similar behavior in that JH was only — 2.0 to — 2.1 

 kcal/mole, the energy for binding being mainly contributed by large en- 

 tropy increases (Klotz and Urquhart, 1949 a). The binding of chloride ion 

 to serum albumin gave a JH of only 0.43 kcal/mole and was, therefore, 

 very little dependent on temperature (Scatchard et al., 1950 a). No effect 

 at all of temperature on the binding of thiocyanate to serum albumin 

 could be found (Scatchard et al., 1950 b). The enthalpies of binding are 

 uniformly small and this applies as well to the binding of aromatic sulfo- 



