Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.
The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.
Chen
B J
BJ
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
Yuan
A I
AI
Wang
D
D
Feinman
R D
RD
D006801:Humans; D007700:Kinetics; D008744:Methylamines; D013438:Sulfhydryl Compounds; D013917:Thrombin; D014357:Trypsin; D000511:alpha-Macroglobulins
D008744:Methylamines; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; C027451:methylamine; D014357:Trypsin; D013917:Thrombin