Abstract
The structure of the acylenzyme intermediate in the hydrolysis of the specific spin-label ester substrate methyl N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-tryptophan ate and its fluoro analogs catalyzed by alpha-chymotrypsin (EC 3.4.21.1) has been determined by electron nuclear double resonance (ENDOR) and molecular modeling methods. By a combination of kinetic and cryoenzymological methods, we have established conditions to stabilize the spin-labeled acylenzyme reaction intermediate. Proton ENDOR features specific for the substrate were assigned on the basis of specific deuteration. From the observed ENDOR shifts for protons and fluorines that correspond to principal hyperfine coupling components, the dipolar hyperfine coupling contributions were calculated to estimate electron-nucleus distances. With these dipolar separations as constraints, conformations of the substrate both free in solution and in the active site of alpha-chymotrypsin were determined by molecular graphics analysis. Comparison of the conformation of the bound substrate to that of the free substrate showed that formation of the acylenzyme requires significant torsional alteration in substrate structure. The structural relationships between active-site residues and the substrate in its ENDOR-assigned conformation are examined with respect to the requirements of stereoelectronic rules for formation and breakdown of the acylenzyme species.
Highlights
The salient observation from these ENDOR studies that the substrate in its acylenzyme form is significantly altered from the conformationsfound in solution is directly relevant to assessment of the molecular basis of substrate recognition by proteolytic enzymes
The active sites of proteolytic enzymes are composed of multiple subsites, each accommodating a residue of an oligopeptide substrate, as illustrated for CKCTin Fig. 8
It is expected that oligopeptide substrates exist in multiple conformations in solution, as does the spin-labeled substrate employed in this investigation
Summary
Dept. of Biochemistry and Molecular Biology, The University of Chicago, CummingsLife. EPR and ENDOR-Spectra were recorded with an X-band Bruker. I. SL methyl L-tryptophanate solution in the EPR tube maintained a t 0 "C, a small aliquot of substrate was injected through a plastic catheter. SL methyl L-(c3-F)tryptophanate substrate and enzyme using the catheter, samples were incubated at 0 "C for different lengths of time, and the reaction was quenched by freezingin liquid nitrogen. SL methyl L-(q2-F)tryptophanate enzyme in EPR sample tubes were 2.1 x and 2.8 x M, respectively, in asolvent mixture of zH20/[2H,]Me2S0(928, v/v). SL ( 13C)methyl L-tryptophanate were stored in liquid nitrogen until use. V. SL methyl L-(a2-H)tryptophanate structed from x-ray-definedmolecular fragments (33, 34) as described previously (6). SL methyl D, L-(LH8)tryptophanate the Brookhaven Protein Data Bank (File 2CHA) (18).Molecular modeling was camed out with the use of the programs FRODO (35), IN-.
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