The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.

Mechanisms of spectral shifts in lobster carotenoproteins. The resonance Raman spectra of ovoverdin and the crustacyanins.

Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin upon binding to the carotenoproteins, ovoverdin and alpha-,beta- and gamma-crustacyanins, from the lobster Homarus americanus. Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to lambdamax at 605 +/- 25 nm, are essentially the same. The large red shift in lambdamax for the crustacyanins compared to free astaxanthin (lambdamax 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up pi electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105,000, 95,000 and 78,000 which are not linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each bound at a different site. The spectral characteristics of the 460 nm site suggest a rigid hydrophobic environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712). The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.

Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.

Preferred conformers and photochemical (lambda > 200 nm) reactivity of serine and 3,3-dideutero-serine in the neutral form.

A systematic investigation of the conformational potential energy surface of neutral serine [HOCH2CHNH2COOH] and 3,3-dideutero-serine [HOCD2CHNH2COOH] was undertaken, revealing the existence of 61 different minima. The structures and vibrational spectra of the most stable conformers, which were estimated to have relative energies within 7 kJ mol(-1) and account for ca. 93% of the total conformational population at room temperature, were calculated at both the MP2 and DFT/BLYP levels of theory with the 6-311++G(d,p) basis-set and used to interpret the spectroscopic data obtained for the compounds isolated in low-temperature inert matrixes. The assignment of the main spectral infrared features observed in the range 4000-400 cm(-1) to the most stable conformers of serine was undertaken. In addition, UV irradiation (lambda > 200 nm) of the matrix-isolated compounds was also performed, leading to decarboxylation, which was found to be strongly dependent on the conformation assumed by the reactant molecule.

Calpha-H bond-stretching frequency in alcohols as a probe of hydrogen-bonding strength: a combined vibrational spectroscopic and theoretical study of n-[1-D]propanol.

The sensitivity of the nu(C)()alpha(-)(H/D) vibrational stretching frequency to hydrogen bonding in alcohols is examined by infrared and Raman spectroscopy, supported by DFT(B3LYP)/6-311++G(d,p) calculations. The model compound studied is (R,S)-n-[1-D]propanol. It is shown that the nu(C)()alpha(-)(H/D) mode can be successfully correlated with the hydrogen-bond strength in a given solvent, provided the O-H group involved in the hydrogen bond is not acting simultaneously as a hydrogen-bond donor and acceptor. In addition, a detailed analysis of the spectroscopic features observed in both the nu(O)(-)(H) and nu(C)()alpha(-)(H/D) spectral regions of the spectra of n-propanol and (R,S)-n-[1-D]propanol, in a series of different experimental conditions, which include the matrix-isolated compound (in argon matrix), pure liquid and low-temperature glassy states, and solution in different solvents, is undertaken. This permits the contribution of the different conformers of the studied compounds to be assigned to the bands observed in the nu(O)(-)(H) and nu(C)(-)(H) spectral regions.

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.

Alpha-helix dipoles and catalysis: absorption and Raman spectroscopic studies of acyl cysteine proteases.

Raman and absorption spectroscopic data are combined with the deacylation rate constants for a series of acyl cysteine proteases to provide insight into the role of alpha-helix dipoles in rate acceleration. The Raman spectra, obtained by Raman difference spectroscopy, of (5-methylthienyl)acryloyl adducts with papain, cathepsins B and L, and two oxyanion hole mutants of cathepsin B (Q23S and Q23A) show that extensive polarization throughout the pai-electron chain occurs for the bound acyl group in the active sites. A similar result is obtained using the specific chromophoric substrate ethyl 2-[(N-acetyl-L-phenylalanyl)amino]-3-(5-methylthienyl)acrylate. By using 13C = O substitution it is possible to detect the acyl C = O stretching frequency, vC = O, for each acyl enzyme. A correlation between vC = O and log k3, where k3 is the deacylation rate constant, is found where vC = O increases with increasing reactivity. This is exactly the opposite sense to the relationship found for a series of acyl serine proteases [Carey & Tonge (1995) Acc. Chem. Res. 28, 8]. The opposite trend in the direction of the correlation for the acyl cysteine proteases is ascribed to the strong electron polarizing forces in the active site, due principally to the active-site alpha-helix dipole, giving rise to canonical (valence bond) forms of the acyl group which change the hybridization about the C = O carbon atom. A correlation is also observed between the absorption maximum, lambda max, and log k3 for each acyl cysteine protease. As the deacylation rate increases, 214-fold across the series, lambda max red-shifts from 367 to 384 nm. It is proposed that differential interactions between the active site's alpha-helix dipole and the acyl chromophore give rise to the observed changes in lambda max, with the red shift being caused principally by interactions with the excited electronic state, which has a high degree of charge separation, and the dipole. Similar interactions between the dipole and the negatively charged tetrahedral intermediate, which resembles the transition state, are proposed as the source of differential rates in deacylation. It is interesting to note that similar energies are operating in both cases. A shift in lambda max from 367 to 384 nm corresponds to a change in electronic absorption transition energies of 3.2 kcal/mol and a change of deacylation rate constants of 214-fold also corresponds to a change of activation energies of 3.2 kcal/mol.

Neoprontosil binding to carbonic anhydrase. Reasonance Raman and other studies on the ionization behavior of the sulfonamide.

Alkalimetric, spectrophotometric, NMR, and resonance Raman titrations are reported for the sulfonamide Neoprontosil in aqueous solution. An assignment of the magnetic resonance peaks for each of the Neoprontosil protons has been made. Neoprontosil is shown to have two "coupled" iity of the microscopic pKs for these two groups precludes spectroscopic characterization of the separate -SO2NH2, -O- or -SO2NH-, -OH species. For this reason, no conclusion can be drawn on the ionization state of the drug when bound to carbonic anhydrase. The resonance Raman spectrum of Neoprontosil bound to human carbonic anhydrase B at pH 9.5 shows a shift in the intense -N=N- stretching mode from 1414 (free) to 1407 cm- (bound), suggesting that a slight conformational change about the -N=N- single bond linkages occurs upon binding.

Molecular structure of 5-methyl thiophene acryloyl ethyl thiolester: a vibrational spectroscopic and density functional theory study.

Enzyme-substrate intermediates involving the acyl group 5-methyl thiophene acryloyl (5-MTA) bound to the active site of an enzyme via a sulfur or selenium atom have been characterized by Raman spectroscopy (e.g., J. D. Doran and P. R. Carey, Biochemistry 1996, 35, 12495-12502, and M. J. O'Connor et al., J Amer Chem Soc 1996, 118, 239-240). Raman difference spectra reveal the Raman spectrum of the acyl group in the active site and, in turn, these can be used to probe acyl group conformation and active site forces and interactions. In order to improve the understanding of the relationship between conformational states and vibrational spectra of 5-MTA thiolesters, calculations based on a density functional theory analysis are undertaken for 5-methyl thiophene acryloyl ethyl ester. The calculations provide the precise geometries and energies of rotomers of 5-MTA ethyl thiolester involving rotational isomerism about the C--C single bonds flanking the ethylenic linkage and the S--C bond linking the ethyl group to the sulfur atom. The calculations also provide the vibrational spectrum for each conformer and these predictions are compared with the experimental Raman an IR data for the thiolester in carbon tetrachloride. Modes are identified that can act as conformational markers for isomerism about the C--C and S--C2H5 single bonds. These findings are used to identify the two conformational states giving rise to the Raman spectrum of the 5-MTA-S-enzyme formed by the viral cysteine protease HAV-3C.

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)

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.

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters.

The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.

Comparison of the kinetics of the papain-catalyzed hydrolysis of glycine- and alanine-based esters and thiono esters.

The kinetic constants for the papain-catalyzed hydrolysis of a series of substrates with glycine or alanine in the P1 position are discussed. The substrates have N-benzoyl, N-(p-nitrobenzoyl), N-(beta-phenylpropionyl), or N-(methyloxycarbonyl)phenylalanine attached to the P1 moiety, and kinetic constants are obtained for both esters and thiono esters. The results for the hydrolysis of esters can be readily interpreted in terms of the known specificity of papain. For any glycine ester the change in kcat/Km upon substituting C=S for C=O or upon substituting an alpha-CH3 group is minimal. However, upon making both these substitutions, i.e., going from a glycine ester to an alanine thiono ester substrate, larger changes are seen for this ratio. Data for N-benzoyl- and N-(beta-phenylpropionyl)glycine and -alanine methyl thiono esters show that k2 is the parameter most affected by the double C=S and alpha-CH3 substitution. A further conclusion is that the deacylation rate constants for any pair of glycine and alanine dithioacyl papains are similar; e.g., for the intermediates based on the "good" substrates PheAla and PheGly k3 differs by only 20%. This is a surprising finding in light of the very different conformations and interactions of the bound acyl groups revealed by resonance Raman spectroscopy and raises the possibility that specific stereochemical effects, such as the oxyanion hole and general base catalysis, are not operating in the hydrolysis of dithioacyl papains.

Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain.

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate). Thus, in this sense the stabilization of the thioimidate does not reflect a similarity in structure between the bound thioimidate and the transition state.

Conformational states of N-acylglycine dithioesters in solution: resonance Raman studies of isotopically substituted models for enzyme-substrate complexes.

Resonance Raman (RR) and FTIR spectroscopic studies, taken with X-ray crystallographic data, are used to define the three major conformational states of N-acylglycine dithioesters in solution and to set up spectra-structure correlations. Importantly, each conformer has a characteristic RR signature, and thus the RR spectrum can be used to follow conformational events within dithioester enzyme-substrate intermediates. The signatures are further defined in the present work by the synthesis and spectroscopic characterization of 13C and 15N derivatives of N-benzoylglycine ethyl dithioester and N-(beta-phenylpropionyl)glycine ethyl dithioester. The observed isotope shifts offer insight into the normal mode character of the RR bands and provide standards with which to compare the shifts in the corresponding enzyme-substrate intermediates.

Relaxed and perturbed substrate conformations in enzyme active sites: evidence from multichannel resonance raman spectra.

A diode array based multichannel Raman spectrometer has made it possible to record complete, high quality, resonance Raman (RR) spectra of enzyme-substrate intermediates. The intermediates are dithioacylpapains in which the acyl group is either N-benzoylglycine or N-(beta-phenylpropionyl)glycine. RR data are reported for the unlabeled dithioacylpapains as well as for the intermediates labeled separately with ND, 15N, and 13C = S in the glycine residue. Comparison of the results for the dithioacylpapains with that of the corresponding labeled glycine ethyl dithioesters [Lee, H., Storer, A. C., & Carey, P. R. (1983) Biochemistry (preceding paper in this issue)] leads to the conclusion that for both substrates in the active site the dihedral angles in the glycine NH-C-C(= S) linkages assume an essentially relaxed type B conformation. Similarly, there is no evidence for distortion about the C(= O)-NH peptide bond which links the P1 and P2 sites on the substrate. However, for the N-benzoylglycine case there is evidence for some conformational distortion in the -S-C-C cysteine linkages. The present data favor a single homogeneous conformational population about the substrates' NH-C-C(= S) bonds in the native dithioacylpapains. However, below pH 3.0 the dithioacyl enzymes denature and the RR spectra of the 13C = S substituted species confirm that the conformational population reverts to the mixture of conformers A and B found for the corresponding ethyl dithioesters in solution.