38 



1. lODOACETATE AND lODOACETAMIDE 



high sensitivity of this enzyme when isolated, the situation may be different 

 in intact muscle, and the effects of iodoacetate in vivo are likely to depend 

 on a variety of factors. 



The alkylation of creatine kinase by iodoacetate and iodoacetamide is 

 not altered by variation of the pH from 6 to 9 but is sensitive to changes 

 in the ionic strength (Fig. 1-2) (Rabin and Watts, 1960; Watts, 1963; Watts 



0.05 



IONIC STRENGTH - 



Fig. 1-2. Effects of ionic strength on the rates 

 of reaction of iodoacetate (0.5 mM) and iodo- 

 acetamide (0.1 mM) with creatine kinase at pH 

 9 and 25°. (From Rabin and Watts, 1960.) 



et al., 1960). Alkylation of a single SH group is sufficient to inactivate the 

 enzyme and possibly a positive charge near this group is responsible for 

 the ionic strength effect with iodoacetate. The reaction with iodoacetamide 

 is not affected by ionic strength, proving that it is not just a configurational 

 change of the enzyme (Watts and Rabin, 1962). The enzyme is protected 

 against iodoacetate by creatine-P and ATP as long as Mg++ is present; 

 neither of the substrates alone nor Mg++ protects, indicating a Mg-complex 

 as the true substrate. The SH group at the active site is pictured as lying 

 adjacent to a histidine residue, with which it forms a hydrogen bond, this 

 explaining to some extent the lack of pH effects. Using an iodide electrode 

 to determine the release of iodide during reaction of the SH groups, the 

 rate of inactivation was found to parallel the rate of carboxymethylation 

 by iodoacetate (Fig. 1-3) (Watts et al., 1961). It may well be that creatine 

 kinase can exist in different forms, inasmuch as Watts et al. (1962) found 

 that substrates protected only one SH group in one sample but protected 

 both of the SH groups in another. Another complication is introduced by 

 the observation of Mahowald et al. (1962) that reaction of the kinase with 



