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Substrate-induced Changes in Protease Active Site Conformation Impact on Subsequent Reactions with Substrates*

By 潘) Rong Pan(荣, 张雪) Xue-Jing Zhang(晶, 张子) Zi-Jian Zhang(剑, 周) Yuan Zhou(园, 田维) Wei-Xi Tian(熙 and 赫荣) Rong-Qiao He(乔


Enzymatic catalysis of biochemical reactions is essential to all living systems. The “lock and key” and “induced fit” models were early contributions to our understanding of the mechanisms involved in the reaction between an enzyme and its substrate. However, whether a given substrate-induced conformation is rigid or remains flexible has not yet been determined. By measuring the enzyme activity and intrinsic fluorescence of a nonspecific Eisenia fetida protease-I with different chromogenic substrates, we show that in subsequent reactions of protease with substrates, both the “lock and key” and “induced fit” mechanisms are used depending on the degree of conformational change required. Chromozym-Th- or chromosym-Ch-induced protease conformations were unable to bind chromozym-U. The chromosym-U-induced protease conformation remained flexible and could be further induced by chromozym-Th and chromozym-Ch. When low concentrations of guanidine HCl were used to disturb the conformation of the enzyme, only small changes in intrinsic fluorescence of the chromozym-Th-induced protease were detected, in contrast to the native enzyme whose intrinsic fluorescence markedly increased. This indicates that the substrate-induced enzyme was relatively rigid compared with the native protease. Utilizing a lock and key mechanism for secondary substrate reactions may have adaptive value in that it facilitates high efficiency in enzymatic reactions

Topics: Enzymology
Publisher: American Society for Biochemistry and Molecular Biology
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Provided by: PubMed Central
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