Conformational Changes in Enzyme Catalysis

Labouesse, Bernard; Havsteen, B.; Hess, George P.

doi:10.1073/pnas.48.12.2137

Cited by 24 publications

(1 citation statement)

References 21 publications

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“…While there is considerable evidence supporting conformational changes in chymotrypsin on adding ligands (Labouesse et al, 1962;Hess et al, 1970;Yapel & Lumry, 1964;Himoe et al, 1967;Henderson, 1970;McClure & Edelman, 1967;Edelman & McClure, 1968), relatively few of these investigations establish the direct involvement of the conformational change in the reaction pathway and even fewer directly link the conformational change to the noncovalent association of enzyme and substrate or inhibitor. The majority of reports of altered CD1 (Hess et al, 1970), ORD and UV 0006-2960/82/0421-4621 $01.25/0 © 1982 American Chemical Society spectra (Labouesse et al, 1962) are concerned with either the now well-characterized alkaline pH transition (Fersht & Requena, 1971a; Kim & Lumry, 1971;Stoesz & Lumry, 1978) or the influence of covalent intermediate formation (Labouesse et al, 1962; Wootton & Hess, 1962; Havsteen & Hess, 1963;Moon et al, 1965).…”

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confidence: 99%

Dynamics of ligand binding to .alpha.-chymotrypsin and to N-methyl-.alpha.-chymotrypsin

Maehler¹,

Whitaker²

1982

Biochemistry

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Ks values for binding of selected substrates, competitive inhibitors, and a noncompetitive inhibitor were found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. The rates and steps of binding of a competitive inhibitor and a noncompetitive inhibitor were also found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. Therefore, N-methyl-alpha-chymotrypsin is an appropriate model for alpha-chymotrypsin in studying the dynamics of the binding of substrates by temperature-jump techniques in aqueous solvents. 2-Toluidinylnaphthalene-6-sulfonate, a noncompetitive inhibitor, bound to alpha-chymotrypsin in a single step with rate constants k1 and k-1 of 3.9 X 10(7) M-1 s-1 and 1.9 X 10(3) s-1, respectively, at pH 5.0 (0.2 M acetate, ionic strength of 0.2). Similar values were obtained for N-methyl-alpha-chymotrypsin and chymotrypsinogen A at pH 5.0 and for alpha-chymotrypsin at pH 7.8 [0.1 M tris(hydroxymethyl)aminomethane-0.03 M CaCl2]. Indole, a competitive inhibitor, bound to alpha-chymotrypsin in a single step at pH 5.0 and 7.8, with k1 and k-1 of 1.8 X 10(7) M-1 s-1 and 7.8 X 10(3) s-1, respectively, at pH 5.0 while proflavin, another competitive inhibitor, bound to alpha-chymotrypsin with two observable steps where k1, k-1, k2, and k-2 were 1.0 X 10(7) M-1 s-1, 7 X 10(2) s-1, 1.0 X 10(3) s-1, and 7 X 10(2) s-1, respectively, at pH 5.0. The specific substrate N-acetyl-L-3,5-dinitrotyrosine ethyl ester bound to N-methyl-alpha-chymotrypsin at pH 5.0 in three observable steps where k1, k-1, k2, k-2, k3, and k-3 were 3.7 X 10(7) M-1 s-1, 6.2 X 10(4) s-1, 1.2 X 10(3) s-1, 3.5 X 10(2) s-1, 3 X 10(2) s-1, and 4 X 10(2) s-1, respectively. Preliminary data indicated that the third step of this reaction is probably absent when Met192 of N-methyl-alpha-chymotrypsin is oxidized to methionine sulfoxide. These results confirm the validity of data obtained from reactions at subzero temperatures in 65% dimethyl sulfoxide in indicating multiple steps in the binding of substrates to alpha-chymotrypsin. The methodology described should make it possible to measure quantitatively the contribution of the binding process to enzyme catalysis (the Circe effect).

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