Infrared Spectroscopy of EnzymeReaction Intermediates

Abstract

The serine proteinase mechanism has been studied using a wide range of techniques over many years and is now well understood in terms of the formal chemical changes that occur on the reaction pathway. At the atomic level our understanding is less secure in that available techniques are unable to define interactions such as hydrogen bonding with sufficient accuracy. Atomic interaction is strongly dependent upon separation distances and these need to be measured either directly or indirectly in the dynamic reacting system. Infrared spectroscopy has been applied to the study of chymotrypsin acylenzyme reaction intermediates with the aim of measuring, albeit indirectly, the strength of hydrogen bonding in the oxyanion hole catalytic device. These measurements have been successful with moderately specific substrates but there is a long way to go in terms of improved time-resolution. It is tentatively proposed that tetrahedral intermediates accumulate at high pH. This is, we believe, the first report of the relatively direct observation of this phenomenon in any reacting ester system, chemical or enzymic. The approach used with the serine proteinases has been applied to studies of the transpeptidase of Streptococcus pneumoniae PBP2x. We have shown that the acylenzyme formed from benzylpenicillin hydrogen bonds most strongly in the oxyanion hole. This bonding, in contrast to serine proteinases and β-lactamases where the interaction facilitates catalysis, serves to stabilise the ester intermediate as required for effective antibiotic action. We see this as a good example of ‘Nature knows Best’ since semisynthetic antibiotics, designed to be resistant to hydrolysis by β-lactamases, hydrogen bond more weakly and the acylenzymes hydrolyse more rapidly.

Local Structure of β-Hairpin Isotopomers by FTIR, 2D IR, and Ab Initio Theory

“…33,34 Isotope labeling of amide groups has proved to be an effective strategy for characterizing local structural features of polypeptides by means of FTIR, 9,35 Raman 36 and most recently 2D IR. 15,17,18,26,27,37,38 The scope of both coherent nonlinear and conventional vibrational spectroscopy and circular dichroism [39][40][41][42] have been greatly enlarged by isotope editing techniques applied to lipids, membranes, peptides and proteins 43 enzyme reaction intermediates 44 and even shifting the IR spectra of whole proteins. 45 Isotope substitution has always been a successful strategy of IR spectroscopy and the applications to biological systems are a natural development 46 whose use is growing.…”

Infrared Difference Spectroscopy as a Physical Tool to Study Biomolecules

“…An infrared spectrum contains a wealth of information on a molecule and its environment, such as bond lengths, bond strengths (22,(48)(49)(50)(51)(52)(53)(54)(55)(56), conformational freedom (5,(57)(58)(59)(60)(61)(62), and hydrogen bonding (63)(64)(65)(66)(67)(68)(69)(70)(71)(72). Anything that changes the electron distribution of the bonds or the environment will change the vibrational frequency and therefore appear in the infrared spectrum.…”

Extended-spectrum β-lactamases: structure and kinetic mechanism

“…Indirect experimental evidence of significant changes in protein conformation has been obtained from reactions in solution, particularly from spectroscopic and stability measurements [1,2]. For example, extensive hydrogen‐to‐deuterium exchange with the deuterated solvent during the deacylation reaction of TEM‐3 was detected by time‐resolved Fourier‐transform infrared spectroscopic studies [3], suggesting that there must be a substantial structural change during the deacylation process that allows access to the core of the protein. As yet, there is little evidence of such changes from X‐ray crystallographic studies of reactions occurring with the crystallised protein, or from proteins crystallised after reaction with inhibitors [4–6].…”

“…As yet, there is little evidence of such changes from X‐ray crystallographic studies of reactions occurring with the crystallised protein, or from proteins crystallised after reaction with inhibitors [4–6]. This dichotomy leads to an inadequacy in describing the mechanism of β‐lactamases and necessitates further investigation of these reactions using techniques such as nuclear magnetic resonance [7] and infrared spectroscopy [3], which can generate time‐resolved structural information from solution reactions.…”