Kinetics of the Reaction of α-Chymotrypsin with Diisopropyl Fluorophosphate, with Measurements of Hydrogen Ion Release, Enzyme Inhibition, and Spectral Changes of Enzyme at 290 mµ

Moon, Aeri; Sturtevant, Julian M.; Hess, George P.

doi:10.1016/s0021-9258(18)97046-8

Cited by 41 publications

(14 citation statements)

References 32 publications

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“…33 Consider the reaction mechanism proposed by Moon, Sturtevant, and Hess where an initial complex is formed at a rate near diffusional, followed by a conformational change to produce a second complex. 34…”

Section: Resultsmentioning

confidence: 99%

“…Using eq 12 and the data from Figure 4, the ratio of relaxation time differences (eq 31) is equal to (94.1)2(1.0 + ( )2) = (56.4)2(1.0 + 2.78( )2) (34) The solution to eq 34 is ( )2 = 0.23 (35) can then be obtained by substituting eq 35 into eq 36 Assuming Kn = 2.0 X -2 M,26•26 the solution to eq 36 is T = 5.9 X 10-3 (37) Hence, k-1 = 1.7 X 102 sec-1 ki = 8.5 X 103 M~l sec"1 (38) and = 14 Hz. The agreement with the results presented in Tables I and II is satisfactory.…”

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confidence: 99%

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Application of transient nuclear magnetic resonance methods to the measurement of biological exchange rates. Interaction of trifluoroacetyl-D-phenylalanine with the chymotrypsins

Sykes¹

1969

J. Am. Chem. Soc.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Application of transient nuclear magnetic resonance methods to the measurement of biological exchange rates. Interaction of trifluoroacetyl-D-phenylalanine with the chymotrypsins

Sykes¹

1969

J. Am. Chem. Soc.

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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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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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“…A and A' are vinyl hydrogens and X and X' are bridgehead hydrogens; in hertz. 6 P. Laszlo and P. von R. Schleyer, /. Amer.…”

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confidence: 99%

“…Rate Ratios (Unsaturated-Saturated) of Related " kM used is a calculated unassisted rate for 15.13 1 Reference 16. c Reference 10. d Reference 2 6. Reference 4.…”

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confidence: 99%

Solvolytic reactions of endo-bicyclo[3.2.1]oct-6-en-8-yl tosylate

Hess¹

1971

J. Am. Chem. Soc.

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The rates of solvolysis of endo-bicyclo[3.2.1]oct-6-en-8-yl tosylate have been determined in acetic acid and ethanol-water mixtures. Product studies indicated that only products with the parent structure were formed. Both the results of product studies and interpretation of rate data support a significant amount of anchimeric assistance by the carbon-carbon double bond.the known ezzz/o-bicyclo[3.2.1]octan-8-ol.10 The nmr spectrum of 9 had absorptions at 4.13 (m, 2, CH=CH), 5.25 (t, 1, CHOAc), 7.48 (m, 2, bridgehead), 7.94 (s, 3, CH3), 8.20-9.08 (general absorption, 6, (CH2)3). At normal sweep width the vinyl region of 9(5) The synthesis and solvolytic behavior of a related compound, endobicyclo[3.2.1]octa-2,6-dien-8-yl tosylate, has been reported: G. Klump,

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Kinetics of the Reaction of α-Chymotrypsin with Diisopropyl Fluorophosphate, with Measurements of Hydrogen Ion Release, Enzyme Inhibition, and Spectral Changes of Enzyme at 290 mµ

Cited by 41 publications

References 32 publications

Application of transient nuclear magnetic resonance methods to the measurement of biological exchange rates. Interaction of trifluoroacetyl-D-phenylalanine with the chymotrypsins

Application of transient nuclear magnetic resonance methods to the measurement of biological exchange rates. Interaction of trifluoroacetyl-D-phenylalanine with the chymotrypsins

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

Solvolytic reactions of endo-bicyclo[3.2.1]oct-6-en-8-yl tosylate

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