VARIATION OF EXZYME INHIBITION WITH pH 701 



2.17 units here and either abolish the inhibition or make it essentially 

 complete, depending on the sign of the charge. These calculations are, of 

 course, very approximate and nothing is known of the value of iv for any 

 enzyme. It is probably valid to conclude that the p^, can be shifted sig- 

 nificantly by a change in pH, either through interactions with vicinal 

 groups or with the net protein charge. 



A very interesting observation, which bears on the present problem, was 

 reported by Klotz and Ayers (1957). When the group (Me)2N— <p— N=N— 

 <p— Hg— was attached to cysteine, the ^K^ of the dimethylamino group 

 was found to be 3.3, but when it was linked to serum albumin, the ^K^ 

 was 1.8. They interpret this as the result of a "frozen" water structure 

 surrounding the protein polar groups which become less accessible. They 

 speak of the meta structure of the protein, the framework which it imposes 

 on the water in its vicinity, and how this may be altered by pH. On the 

 other hand, in the pH range below 4, serum albumin must be strongly 

 positively charged. There are nearly one hundred ionizable groups that 

 should be positively charged in this region, and below a pH of 4 the net 

 positive charge must increase rapidly due to the protonation of the car- 

 boxyl groups. From the curve for p^, in Fig. 14-15, a shift of 1.5 units would 

 be brought about by around thirty-five positively charged groups but 

 since iv = 0.021 for serum albumin, a total of eighty-six such groups 

 would be necessary, which is not unreasonable. Therefore, without min- 

 imizing the importance of bound water, it would appear that this result 

 would be attributed to the interaction of the group with the net protein 

 charge. 



Effects of Charged Inhibitors on the pKg's of Groups at the Active Center 



As a charged molecule approaches an ionizing group on an enzyme, the 

 affinity of this group for protons is altered, and this would be expressed 

 as a change of the i:>K^. Thus the combination of an enzyme with charged 

 substrate or inhibitor molecules will cause a shift in the piC^'s of groups at 

 or close to the active center. An experimentally determined jiK^ may refer 

 to the ES or the EI complex and this j)K^ may be significantly different 

 from the Y)K,, of the same group on the free enzyme. This may be important 

 in the identification of the nature of the ionizing group. We have seen that 

 in the Dixon method of plotting, the ionizations of free enzyme and en- 

 zyme complex groups can often be distinguished by the direction of the 

 inflections in the curves. 



The formation of comi)lexes between fumarase and fumarate or malate 

 has been shown to alter the p^^'s of at least two enzyme groups (Massey 

 and Alberty. 1954; Frieden and Alberty, 1955; Alberty, 1956 a). The 

 results are summarized in Table 14-3 and it is seen that shifts of + 0.4 

 to +1.6 were usually demonstrable. The one discrepancy, the reduction 



