Peter J. Tonge

A quick method for purifying bile salt-activated lipases

A simple, one step chromatography procedure of general utility for purifying bile salt-activated lipases (BAL; cholesterol esterases) using a cholate affinity column is described. A key feature of the method is the use of a taurocholate concentration gradient in the eluent. The method has been used successfully to purify bovine and porcine BAL from commercial crude sources and BAL from human milk whey.

Resonance Raman and absorption spectroscopic characterization of the chemically engineered enzyme thiolsubtilisin; comparison with a natural thiolenzyme

Abstract The serine protease subtilisin Carlsberg has been converted to the cysteine protease thiolsubtilisin by chemically converting the active site Ser-221 to cysteine. The present experiments have been designed to explore the differences in the active site properties of thiolsubtilisin and papain - a natural cysteine protease. Resonance Raman and electronic absorption studies for the enzyme—substrate intermediates involving the acyl groups 5-methylthienylacryloyl and furylacryloyl have been used to probe the electrical effects of thiolsubtilisins active site on the groups delocalised π-electron systems. Unlike papain, thiolsubtilisin does not give rise to highly polarized π-electron systems in the bound substrates, characterized by low frequency νCue5fbC modes and red shifted λmaxs. However, for the 5-methylthienylacryloyl intermediate there is evidence for a minor photoinduced population which does have a polarized π-electron system. The results can be explained by differential binding of the substrate with respect to the α-helix dipole found in the active sites of thiolsubtilisin and papain.

Resonance Raman spectroscopic and kinetic consequences of a nitrogen ... sulphur enzyme-substrate contact in a series of dithioacylpapains.

The resonance Raman (RR) spectroscopic, conformational, and kinetic properties of six dithioacylpapain intermediates have been examined. Five of the intermediates are of the form N-(methyloxycarbonyl)-X-glycine-C(= S)S-papain, where X is L-phenyl-alanine, D-phenylalanine, glycine, L-phenylglycine, or D-phenylglycine. The sixth intermediate is N-phenylacetyl-glycine-C(= S)S-papain. Throughout the series there is an approximately 50-fold variation in kcat, the rate constant for deacylation, and a 1750-fold variation in kcat/KM. Existing RR spectra structure correlations allow us to define the torsional angles in the NH-CH2-C(= S)-S-CH2-CH fragment of the functioning intermediates. The values of these angles for each bound substrate appear to be very similar, with the substrates assuming a B-type conformer such that the nitrogen atom of the P1 glycine residue is cis to the thiol sulphur atom of cysteine-25. For each intermediate, the C(= S)S-CH2CH torsional angle is approximately -90 degrees, whereas for the SCH2-CH torisonal angle the cysteine-25 thiol sulphur (S) and cysteine-25 C alpha hydrogen (H) atoms are approximately trans. The three acyl-enzymes with the lowest catalytic rate constants, viz. N-(methyloxycarbonyl)-glycine-glycine-, N-(methyloxycarbonyl)-L-phenylglycine-glycine-, or N-(phenylacetyl)-glycine-dithioacylpapains, have atypical RR spectra in that they show a feature of medium intensity in the 1,085-cm-1 region. This band is sensitive to NH to ND exchange of the P1 glycine residues (-NH-) function and, thus, the corresponding mode involves an excursion of the NH hydrogen. It is hypothesized that the high intensity is due to a particularly strong interaction between the P1 glycine nitrogen atom and the thiol sulphur of cysteine-25, which also has the effect of retarding deacylation, because the nitrogen . . . sulphur contact has to be broken in the rate-determining step.

Time resolution of events in an enzyme's active site at 4 K and 300 K using resonance Raman spectroscopy

Resonance Raman spectra for enzyme-substrate intermediates of the type R(C are reported in solution at 300 K and down to 4 K in ice matrices. Analysis reveals that the overall conformation of the substrate-enzyme bonds in the active site remains the same in the range 300 - 4 K but there are important minor spectral changes. Some of these provide access to information on dynamical fluctuations occurring in the active site. Evidence for closely lying fluctuating protein states driving fluctuations in the structure of the bound substrate is discussed. 2.