Milner-White E J, Kelly I D
Biochem J. 1976 Jul 1;157(1):23-31. doi: 10.1042/bj1570023.
Protection against inhibition of creatine kinase by iodoacetamide is measured by the decrease in the rate constant for the inhibition reaction; A mixture of purified substrates at equilibrium protects quite strongly when all the components of the mixture are nearly saturating. The protection by substrates 'working' in the forward direction only (from creatine and MgATP) was measured by carrying out the experiment rapidly at low concentrations of the enzyme; by varying the concentration of substrate it was found that the amount of protection when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the rate constant of the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. It is already known that the complex creatine-NO3--MgADP, which is considered to be either a transition-state analogue or an analogue of an intermediate in the reaction pathway, protects fully against iodoacetamide, whereas creatine and MgADP alone, or together without NO3-, do not protect. This suggests that the degree of protection by the working enzyme represents the proportion of enzyme molecules that have a conformation complementary to a creatine-PO3-MgADP intermediate.
通过抑制反应速率常数的降低来测定对碘乙酰胺抑制肌酸激酶的防护作用；当混合物的所有组分几乎都处于饱和状态时，处于平衡状态的纯化底物混合物具有很强的防护作用。仅通过正向反应（由肌酸和MgATP）“起作用”的底物的防护作用，是在低酶浓度下快速进行实验来测定的；通过改变底物浓度发现，当正向反应的底物饱和时，防护量约为80%（100%的防护意味着抑制反应速率常数的值为零）。比较了Ca2+和Mg2+的影响。已知肌酸 - NO3--MgADP复合物，被认为是过渡态类似物或反应途径中中间体的类似物，能完全防护碘乙酰胺，而单独的肌酸和MgADP，或没有NO3-时两者一起，则没有防护作用。这表明起作用的酶的防护程度代表了具有与肌酸 - PO3 - MgADP中间体互补构象的酶分子的比例。
