Biomedical subjects

P J Tonge

Publications and source records attributed to P J Tonge.

24 records · Page 2Linked to original sources

PubMed1994

Electronic rearrangement induced by substrate analog binding to the enoyl-CoA hydratase active site: evidence for substrate activation.

A series of alpha,beta unsaturated CoA thiol esters have been characterized spectroscopically when they form noncovalent complexes at the active site of enoyl-CoA hydratase. The UV spectra of all of the thiol esters display significant red shifts when the esters are bound to the crotonase active site. The red shift increases with the ability of a para substituent of substituted cinnamoyl-CoA thiol esters to donate electrons by resonance. The affinity of the substituted cinnamoyl-CoA thiol esters is enhanced by electron-donating substituents, with the slope of the log of the ratio of the inhibition constants versus sigma p+ being near unity. Affinity is also increased by either para or meta electron-withdrawing substituents, suggesting that the enzyme stabilizes a partial positive charge at C-3. Binding to crotonase was shown to decrease the shielding of [3-13C,3-2H]cinnamoyl-CoA by +3.2 ppm, consistent with an increased partial positive charge at C-3. The Raman spectra of cinnamoyl-CoA bound at the crotonase active site similarly reflect the significant electronic ground state changes in the pi electronic structure of the bound substrate. These data show that a major rearrangement of electrons occurs in the acryloyl portion of the cinnamoyl group upon binding, while only a minor perturbation occurs to the distribution of electrons in the phenyl ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗

PubMed1992

Forces, bond lengths, and reactivity: fundamental insight into the mechanism of enzyme catalysis.

Comparison of spectroscopic, kinetic, and thermodynamic data for a series of functioning acylserine proteases suggests that the observed variation in deacylation rates can be accounted for by changes in the properties of the acyl-enzyme's ground state. The acyl-enzyme's catalytically crucial acyl carbonyl group is probed by resonance Raman spectroscopy. Its spectral frequency is used to gauge both the carbonyl bond length and the strength of hydrogen bonding (originating from groups making up the oxyanion hole) to the carbonyl oxygen atom. As the deacylation rate increases 16,300-fold through the series, a shift in carbonyl frequency, vC = O, of -54 cm-1 corresponds to a carbonyl bond length increase of 0.025 A. The decrease in vC = O is also consistent with an increase in hydrogen bond donor enthalpy of -27 kJ mol-1. Interestingly, this value resembles closely the decrease in activation energy for deacylation through the series, 24 kJ mol-1, demonstrating that the hydrogen bonds to the carbonyl oxygen atom can provide sufficient energy to account for the observed rate accelerations.

Acylation↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1991

Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques.

The acyl carbonyl group of [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin (5MeTA-chymotrypsin) has been investigated by using both resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. The spectrum of the acyl-enzyme carbonyl group has been obtained as a function of pH over the range 3.0-10.0 in the RR experiments and over the range 3.4-7.6 (p2H) in the FTIR experiments. The carbonyl spectral profiles obtained by using FTIR spectroscopy are substantially different from the carbonyl profiles obtained by using RR spectroscopy. The FTIR spectra were obtained by subtracting the spectrum of the free enzyme from that of the acyl-enzyme. Use of the active-site inhibitor phenylmethanesulfonyl fluoride demonstrates that part of the intensity observed in the FTIR spectra of 5MeTA-chymotrypsin is due to a subtraction artifact giving rise to enzyme-associated bands, probably from peptide groups perturbed by substrate binding. The enzyme bands can be removed by subtracting the FTIR spectrum of 13C=O acyl-enzyme from that of 12C=O acyl-enzyme. Additionally, this procedure reveals that one of the acyl-enzyme carbonyl bands observed at 1727 cm-1 using RR spectroscopy is absent in the FTIR acyl-enzyme spectrum. However, a feature near 1720 cm-1 can be induced in the FTIR spectrum by actinic light in the near-UV region. Thus, it is proposed that the 1727 cm-1 RR carbonyl band results from a population of acyl-enzymes which is generated by exposure to the laser beam during RR data collection. When both the RR and FTIR data are adjusted to remove artifacts, they provide essentially identical carbonyl stretching profiles.

Binding Sites↗

PubMed1990

Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity.

Resonance Raman (RR) spectroscopy has been used to obtain the vibrational spectrum of the acyl carbonyl group in a series of acylchymotrypsins and acylsubtilisins at the pH of optimum hydrolysis. The acyl-enzymes, which utilize arylacryloyl acyl groups, include three oxyanion hole mutants of subtilisin BPN', Asn155Leu, Asn155Gln, and Asn155Arg, and encompass a 500-fold range of deacylation rate constants. For each acyl-enzyme a RR carbonyl band has been identified which arises from a population of carbonyl groups undergoing nucleophilic attack in the active site. As the deacylation rate (k3) increases through the series of acyl-enzymes, the carbonyl stretching band (vC = O) is observed to shift to lower frequency, indicating an increase in single bond character of the reactive acyl carbonyl group. Experiments involving the oxyanion hole mutants of subtilisin BPN' indicate that a shift of vC = O to lower frequency results from stronger hydrogen bonding of the acyl carbonyl group in the oxyanion hole. A plot of log k3 against vC = O is linear over the range investigated, demonstrating that the changes in vC = O correlate with the free energy of activation for the deacylation reaction. By use of an empirical correlation between carbonyl frequency (vC = O) and carbonyl bond length (rC = O) it is estimated that rC = O increases by 0.015 A as the deacylation rate increases 500-fold through the series of acyl-enzymes. This change in rC = O is about 7% of that expected for going from a formal C = O double bond in the acyl-enzyme to a formal C-O single bond in the tetrahedral intermediate for deacylation. The data also allow us to estimate the energy needed to extend the acyl carbonyl group along its axis to be 950 kJ mol-1 A-1.

Acylation↗

PubMed1989

Direct observation of the titration of substrate carbonyl groups in the active site of alpha-chymotrypsin by resonance Raman spectroscopy.

By use of resonance Raman (RR) spectroscopy, the population of the reactive carbonyl group in active acylchymotrypsins has been characterized and correlated with acyl-enzyme reactivity. RR spectra have been obtained, with a flow system and 324- and 337.5-nm excitation, at low and active pH for six acylchymotrypsins, viz., (indoleacryloyl)-, (4-amino-3-nitrocinnamoyl)-, (furylacryloyl)-, [( 5-ethylfuryl)-acryloyl]-, (thienylacryloyl)-, and [( 5-methylthienyl)acryloyl]chymotrypsin. These acyl-enzymes represent a 100-fold range of deacylation rate constants. Good RR spectral quality has enabled us to obtain the vibrational spectrum of the carbonyl group at low and active pH in each acyl-enzyme. The measured pKa of the spectroscopic changes in the carbonyl region is identical with that for the deacylation kinetics, showing that the RR carbonyl features reflect the ionization state of His-57. A carbonyl population has been observed in the active acyl-enzymes in which the carbonyl oxygen atom of the reactive acyl linkage is hydrogen-bonded in the active site. The proportion of this hydrogen-bonded population, with respect to other observed non-hydrogen-bonded species, together with the degree of polarization of the carbonyl bond, as monitored by vC = 0, has been correlated with the deacylation rate constants of the acyl-enzymes. It is proposed that the hydrogen-bonded carbonyl species is located at or near the oxyanion hole and represents the ground state from which deacylation occurs. An increase in the proportion of the hydrogen-bonded population and an increase in polarization of the carbonyl bond result in an increase in deacylation rate constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗