Substrate-induced changes in protease active site conformation impact on subsequent reactions with substrates

Abstract

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.

FIGURE 1.

12% SDS-PAGE of purified EfP-I.

FIGURE 2.

Activity of EfP-I on a secondary substrate. EfP-I was first reacted with one substrate and then with another, followed by measurement of absorbance at 405 nm at 25 °C, CU first, CTH second (A); CTH first, CU second (B); CU first, CCH second (C); CCH first, CU second (D); CTH first, CCH second (E); CCH first, CTH second (F); CTH first, CTH second as a control (G); CU first, CU second as a control (H); and CCH first, CCH second as a control (I).

FIGURE 3.

Changes in the activity of EfP-I induced by the substrates. The first substrate was added to EfP-I and allowed to react to completion. Activity was measured by recording the absorbance of the enzyme reaction at 405 nm. Products and residual substrates were then removed from the incubated samples by four rounds of ultrafiltration (4000 rpm, 25 °C, 20 min). The second substrate was then reacted with the induced enzyme, followed by measurements of absorbance at 405 nm. After the reaction reached completion, products and residual substrates were removed again. The absorbance at 405 nm was measured again in the presence of the first substrate. A, activity of EfP-I^CTH and native EfP-I with CU. B, activity of EfP-I^CTH+CU and native EfP-I with CTH. C, activity of EfP-I^CU and native EfP-I with CTH. D, activity of EfP-I^CU+CTH and EfP-I with CU. E, activity of EfP-I^CCH and native EfP-I with CU. F, activity of EfP-I^CCH+CU and native EfP-I with CCH. G, activity of EfP-I^CU and native EfP-I with CCH. H, activity of EfP-I^CU+CCH and native EfP-I with CU. I, activity of EfP-I^CTH and native EfP-I with CCH. J, activity of EfP-I^CTH+CCH and native EfP-I with CTH. K, activity of EfP-I^CCH and native EfP-I with CTH. L, activity of EfP-I^CCH+CTH and EfP-I with CCH. Error bars represent the S.D.

FIGURE 4.

Conformational changes of EfP-I induced by different substrates. EfP-I was incubated with different substrates CTH (A), CCH (B), and CU (C) at 25 °C for 2 h, and the intrinsic fluorescence (λ_em 330 nm, λ_ex 292 nm) was then measured on a Hitachi F-4500 fluorophotospectrometer. Native EfP-I was used as a control. Error bars represent the S.D.

FIGURE 5.

Conformational changes of EfP-I induced repetitively by different substrates. The effects of different substrates on the conformation of the enzyme were determined by comparing the intensity of the intrinsic fluorescence. To exclude disturbance from the residual substrates and products, each sample was ultrafiltrated before fluorescence was measured. EfP-I, native protease; EfP-I^CCH, enzyme reacted with CCH and ultrafiltrated to remove residual substrates and products; EfP-I^CCH+CCH, enzyme reacted with CCH and ultrafiltrated and then reacted again with CCH and ultrafiltrated; EfP-I^CCH+CU, enzyme reacted with CCH and ultrafiltrated and then reacted again with CU and ultrafiltrated. A, effect of CCH on the conformation of EfP-I^CU. B, effect of CU on EfP-I^CCH. C, effect of CCH on EfP-I^CTH. D, effect of CTH on EfP-I^CCH. E, effect of CTH on EfP-I^CU. F, effect of CU on EfP-I^CTH. EfP-I, EfP-I^CCH+CCH, EfP-I^CU+CU, and EfP-I^CTH+CTH were used as controls. Error bars represent the S.D. G, intrinsic fluorescence of EfP-I^CTH at different concentrations of guanidine hydrochloride (GuHCl), with native EfP-I used as control. H, urokinase-like activity of EfP-I^CTH at different concentrations of guanidine hydrochloride. Error bars represent the S.D.

FIGURE 6.

Proposed mechanism of EfP-I action involving both induced fit and lock and key models. A, EfP-I reacting with the same chromogenic substrate: first by induced fit and subsequently by lock and key. B, EfP-I reacting sequentially with CU, CTH, and CU, undergoing induced fit and then lock and key. C, EfP-I reacting sequentially with CTH, CU, and CTH, undergoing induced fit and then lock and key.