Effect of specificity on ligand conformation in acyl-chymotrypsins

Abstract

I.r. difference spectroscopy combined with 13C and 18O double-isotope substitution was used to examine the ester acyl carbonyl stretching vibration of hydrocinnamoyl-chymotrypsin. A single acyl carbonyl stretching band was observed at 1731 cm-1. This contrasts with previous i.r. and resonance Raman spectroscopic studies of a number of trans-3-arylacryloyl-chymotrypsins which showed two acyl carbonyl stretching bands in the region of 1700 cm-1, which were proposed to represent productive and non-productive conformations of the acyl-enzyme. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin suggests only a single conformation, and the comparatively high frequency of this band implies little or no hydrogen-bonding to this carbonyl group. Enzymic hydrogen-bonding to the acyl carbonyl is believed to give bond polarization and thereby catalytic-rate acceleration. Thus, in view of the apparent lack of such hydrogen-bonding in hydrocinnamoyl-chymotrypsin, it should be the case that this acyl-chymotrypsin is less specific than trans-3-arylacryloyl-chymotrypsins, whereas the opposite is true. It is therefore proposed that there may be a productive acyl carbonyl population of lower stretching frequency for hydrocinnamoyl-chymotrypsin, but that this is too small to be discerned because of either a relatively high deacylation rate or an unfavourable conformational equilibrium. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin is significantly broader than those for trans-3-arylacryloyl-chymotrypsins, indicating that this group is more conformationally mobile and dispersed in the former. This can be correlated with the absence of acyl carbonyl hydrogen-bonding in hydrocinnamoyl-chymotrypsin, and with the much greater flexibility of the saturated hydrocinnamoyl group than unsaturated trans-3-arylacryloyl. This flexibility is presumably the reason why hydrocinnamoyl-chymotrypsin is more specific than trans-3-arylacryloyl-chymotrypsins. Resonance Raman spectroscopy is limited to the non-specific trans-3-arylacryloyl-chymotrypsins because of its chromophoric requirement, whereas i.r. may be used to examine non-chromophoric more specific acyl-enzymes such as hydrocinnamoyl-chymotrypsin. The results presented in this paper suggest that trans-3-arylacryloyl-chymotrypsins are atypical.

Analysis of Pulmonate Mucus by Infrared Spectroscopy

“…. Infrared spectroscopy has been used to examine vertebrate mucopolysaccharides (Barbucci, Magnani, Baszkin, Da Costa, Bauser, Hellwig, Martuscelli, & Cimmino, 1993), proteins (White & Wharton, 1990;Johal, White, & Wharton, 1994), and invertebrate mucus (Watanabe, 1976;Pancake and Karnovsky, 1971), where molluscan pedal mucus was analysed using pressed KBr discs which produced useable IR-spectra between 600 cm -1 and 1800 cm -1 . This produced a much more restricted profile than those described in this paper, and suffered from large-scale water contamination.…”

Discrete steps in sensing of β-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium

“…In both examples, one (or more) acyl-protein intermediates were detected, and the conformational changes that accompanied these reactions were observed (14)(15)(16)(17). Herein we report the application of stoppedflow Fourier-transformed IR spectroscopy (18,19) to the simultaneous observation of the rapid reaction kinetics, the evaluation of the structure of the reaction intermediates, and the interpretation of the induced conformational changes accompanying the reaction of the BlaR1 receptor with a ␤-lactam.…”

“…Such an effect could compromise experiments of this type, if the isotope-edited difference method is not used. Isotope editing specifically removes any effect on the protein that does not directly result from the N-carboxylation status, because the two ''halves'' of the experiment exactly compensate (19).…”

A Dynamic Structure for the Acyl−Enzyme Species of the Antibiotic Aztreonam with the Citrobacter freundii β-Lactamase Revealed by Infrared Spectroscopy and Molecular Dynamics Simulations

“…Knowles has observed, and decisively assigned by using isotope substitution, a band that arises from protein perturbation at 1748 cm -1 in the complex of aldolase with fructose bisphosphate at pD 7.6 (31). We have described a similar observation for the acylenzymes β-phenylpropionyl-and transcinnamoyl-chymotrypsin, which show perturbation band at 1737 and 1736 cm -1 respectively, albeit at low pH (4,32). We have previously argued that such an interpretation is unlikely for frequencies below 1735 cm -1 , and the high frequency bands (1746 cm -1 in the aztreonam spectrum and 1737 cm -1 in the methicillin spectrum) that might be susceptible to such interpretation show different frequencies in the two difference spectra.…”