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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational states of N-acylalanine dithio esters: correlation of resonance Raman spectra with structures

The conformational states of N-acylalanine dithio esters, involving rotational isomers about the RC(=O)NH--CH(CH3) and NHCH(CH3)--C(=S) bonds, are defined and compared to those of N-acylglycine dithio esters. The structure of N-(p-nitrobenzoyl)-DL-alanine ethyl dithio ester has been determined by X-ray crystallographic analysis; it is a B-type conformer with the amide N atom cis to the thiol sulfur. Raman and resonance Raman (RR) measurements on this compound and for the B conformers of solid N-benzoyl-DL-alanine ethyl dithio ester and N-(beta-phenylpropionyl)-DL-alanine ethyl dithio ester and its NHCH(CD3)C(=S) and NHCH(CH3)13C(=S) analogues are used to set up a library of RR data for alanine-based dithio esters in a B-conformer state. (Methyloxycarbonyl)-L-phenylalanyl-L-alanine ethyl dithio ester crystallizes in an A-like conformational state wherein the alanine N atom is nearly cis to the thiono S atom (C=S) [Varughese, K.I., Angus, R.H., Carey, P.R., Lee, H., & Storer, A.C. (1986) Can. J. Chem. 64, 1668-1673]. RR data for this solid material in its isotopically unsubstituted and CH(C-D3)C(=S) and CH(CH3)13C(=S) forms provide information on the RR signatures of alanine dithio esters in A-like conformations. RR spectra are compared for the solid compounds, for N-(p-nitrobenzoyl)-DL-alanine, N-(beta-phenylpropionyl)-DL-alanine, and (methyloxycarbonyl)-L-phenylalanyl-DL-alanine ethyl dithio esters, and for several 13C=S- and CD3-substituted analogues in CCl4 or aqueous solutions. The RR data demonstrate that the alanine-based dithio esters take up A, B, and C5 conformations in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.     

Conformational variability in an enzyme's active site: resonance Raman evidence for different acyl group conformations in N-acylglycine and N-acylalanine dithioacyl papains

It is demonstrated that the vibrational modes associated with the catalytically labile region of N-acylalanine dithioacyl papains undergo a major reorganization compared to the normal modes of corresponding model compounds. Thus, the resonance Raman (RR) spectrum of, e.g., N-benzoylalanine dithioacyl papain and its response to isotopic labeling cannot be understood completely on the basis of the RR spectrum of N-benzoylalanine ethyl dithio ester in one of its known conformational states [detailed in Lee, H., Angus, R. H., Storer, A. C., Varughese, K. I., & Carey, P. R. (1988) Biochemistry (preceding paper in this issue)]. This situation contrasts sharply to that for N-acylglycine dithioacyl papains whose RR spectra closely resemble those of the corresponding N-acylglycine ethyl dithio esters in a conformational state known as conformer B. For the N-acylalanine intermediates two possible causes are put forward to explain the rearrangement of the normal modes. First, the acyl groups based on alanine may bind in papain's active site in a conformation whose torsional angles near the -C(=S)S-group differ markedly from those of characterized model compounds. The second, and presently favored, explanation is that the N-acylalanine moiety is binding in the active site in an A- or C5-like conformation and that, in addition, there is significant vibrational coupling between some of the normal modes of the bound substrate and the normal modes associated with parts of the enzyme in contact with the substrate. The finding that deacylation for N-acylglycine or N-acylalanine dithioacyl papains must proceed from structures which are different is an indication that the mechanism of deacylation may not have strict stereochemical requirements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy

Resonance Raman spectroscopic data provide conclusive evidence for the existence of an acyl-enzyme intermediate during the reaction of a thionoester substrate, N-methyloxycarbonylphenylalanylglycine methyl thionoester (CH3OC(=O)-Phe-NHCH2C(=S) OCH3), with cathepsin B from porcine spleen. The resonance Raman spectrum of CH3OC(=O)-Phe-NHCH2C(=S)S-cathepsin B, where the thiol S is from the active-site cysteine residue, is compared to that of the corresponding papain acyl-enzyme. Within the limits of experimental error (+/-2 cm-1 for peak positions), there are no detectable spectral differences. Since the resonance Raman spectrum is sensitive to the torsional angles in the glycinic bonds and the cysteine linkages, the conformations are identical in those parts of the acyl-enzymes where chemical transformation occurs. A conformational analysis of the model compound CH3OC(=O)-Phe-NHCH2C(=S)SC2H5 demonstrates that the dithioacyl group in both dithioacyl-enzymes is present as a single population of a form known as conformer B. Conformer B is characterized by a small torsional angle about the glycinic NHCH2-CS(thiol) bond such that the nitrogen and S (thiol) atoms are in close contact. This conformer is widespread among the dithioacyl intermediates of plant cysteine proteinases, and it is apparent that the same chemistry is retained in a mammalian cysteine proteinase. Steady-state kinetic parameters are also reported for CH3OC(=O)-Phe-NHCH2C(=S)OCH3 reacting with papain and cathepsin B. The similarity of the Kcat values, 0.53 and 1.15 s-1, for papain and cathepsin B, respectively, provides further evidence for a conserved deacylation process.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

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.     

A resonance Raman study on the reaction intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra of two reaction intermediates of D-amino acid oxidase with substrate analogs were obtained. The reaction intermediates studied were (1) the one in the aerobic oxidative reaction of the enzyme with beta-cyano-D-alanine and (2) the other in the reverse reductive reaction of the enzyme with chloropyruvate and ammonium. Both intermediates are characterized with the charge transfer absorption bands in the long wavelength region extending beyond 600 nm. The RR spectra of the two intermediates excited at 488.0 or 514.5 nm are those of oxidized flavin, which is consistent with our previous assumption that oxidized flavin is involved in these reaction intermediates. Relatively simple RR spectra were obtained for these intermediates with excitation at 632.8 nm which is within the region of the charge transfer bands. The resonance enhancement for the Raman lines around 1585 and 1350 cm-1 for either of the intermediates with excitation in the region of the charge transfer bands suggests that the charge transfer interaction involves the N(5)-C(4a) region extending to the C(10a)-N(1)-C(2) region of the isoalloxazine nucleus. The Raman line at 1657 cm-1 for the intermediate with chloropyruvate and ammonium was assigned to C = N of an imino acid from the isotopic frequency shift upon 15N-substitution. The assignment substantiates our previous conclusion that the intermediate involves an imino acid, alpha-imino-beta-chloropropionate.     

The chromophore structure of the long-lived intermediate of the C128T channelrhodopsin-2 variant

The photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV–vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal–protein linkage.     

Resonance Raman spectroscopy of sensory rhodopsin II from Natronobacterium pharaonis

Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).     

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.     

Raman and surface enhanced Raman spectroscopy of 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide labeled proteins: bovine serum albumin and cytochrome c

2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine serum albumin and cytochrome c at different pH values were prepared and investigated using Raman-resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins in the pH 6.7-11 range were compared to those of the corresponding free species. The SERS spectra were interpreted in terms of the structural changes of the tempyo labeled proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be inactive in the Raman-RR and SERS spectra of the proteins. The alpha-helix conformation was concluded to be more favorable as the SERS binding site of bovine serum albumin. In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.     

Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory

Proper analysis of label distribution in metabolic pathway intermediates is critical for correct interpretation of experimental data and strategic experimental design. While, for example, 13C nuclear magnetic resonance (NMR) spectroscopy is usually limited to the measurement of degrees of 13C enrichment, more information about metabolic fluxes can be extracted from the fine structure of NMR spectra, or molecular weight distributions of isotopomers of metabolic intermediates (measured by gas chromatography-mass spectrometry). For this purpose, rigorous accounting for the contribution of all pathways to label distribution is required, especially contributions resulting from multiple turns of metabolic cycles. In this paper we present a mathematical model developed to analyze isotopomer distributions of tricarboxylic acid cycle (TCA) intermediates following the administration of 13C (or 14C) labeled substrates. The theory presented provides the basis to analyze 13C NMR spectra and molecular weight distributions of metabolites. In a companion paper (Park et al., 1999), the theory is applied to the analysis of several cases of biological significance. Copyright 1999 John Wiley & Sons, Inc.     

Structures of reaction intermediates of bovine cytochrome c oxidase probed by time-resolved vibrational spectroscopy

Structures of reaction intermediates of bovine cytochrome c oxidase (CcO) in the reactions of its fully reduced form with O2 and fully oxidized form with H2O2 were investigated with time-resolved resonance Raman (RR) and infrared spectroscopy. Six oxygen-associated RR bands were observed for the reaction of CcO with O2. The isotope shifts for an asymmetrically labeled dioxygen, (16)O(18)O, has established that the primary intermediate of cytochrome a3 is an end-on type dioxygen adduct and the subsequent intermediate (P) is an oxoiron species with Fe=O stretch (nu(Fe=O)) at 804/764 cm(-1) for (16)O2/(18)O2 derivatives, although it had been long postulated to be a peroxy species. The P intermediate is converted to the F intermediate with nu(Fe=O) at 785/751 cm(-1) and then to a ferric hydroxy species with nu(Fe-OH) at 450/425 cm(-1) (443/417 cm(-1) in D2O). The rate of reaction from P to F intermediates is significantly slower in D2O than in H2O. The reaction of oxidized CcO with H2O2 yields the same oxygen isotope-sensitive bands as those of P and F, indicating the identity of intermediates. Time-resolved infrared spectroscopy revealed that deprotonation of carboxylic acid side chain takes place upon deligation of a ligand from heme a3. UV RR spectrum gave a prominent band due to cis C=C stretch of phospholipids tightly bound to purified CcO.     

Characterization of kinetic and thermodynamic phases in the prefolding process of bovine pancreatic ribonuclease A coupled with fast SS formation and SS reshuffling

In the redox-coupled oxidative folding of a protein having several SS bonds, two folding phases are usually observed, corresponding to SS formation (oxidation) with generation of weakly stabilized heterogeneous structures (a chain-entropy losing phase) and the subsequent intramolecular SS rearrangement to search for the native SS linkages (a conformational folding phase). By taking advantage of DHS(ox) as a highly strong and selective oxidant, the former SS formation phase was investigated in detail in the oxidative folding of RNase A. The folding intermediates obtained at 25 °C and pH 4.0 within 1 min (1S°-4S°) showed different profiles in the HPLC chromatograms from those of the intermediates obtained at pH 7.0 and 10.0 (1S-4S). However, upon prolonged incubation at pH 4.0 the profiles of 1S°-3S° transformed slowly to those similar to 1S-3S intermediate ensembles via intramolecular SS reshuffling, accompanying significant changes in the UV and fluorescence spectra but not in the CD spectrum. Similar conversion of the intermediates was observed by pH jump from 4.0 to 8.0, while the opposite conversion from 1S-4S was observed by addition of guanidine hydrochloride to the folding solution at pH 8.0. The results demonstrated that the preconformational folding phase coupled with SS formation can be divided into two distinct subphases, a kinetic (or stochastic) SS formation phase and a thermodynamic SS reshuffling phase. The transition from kinetically formed to thermodynamically stabilized SS intermediates would be induced by hydrophobic nucleation as well as generation of the native interactions.     

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)     

Semiempirical and Raman spectroscopic studies of carotenoids

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.     

Comparison of the substrate conformations in the active sites of papain, chymopapain, ficin and bromelain by resonance Raman spectroscopy

The resonance Raman spectra of several enzyme-substrate intermediates of papain, chymopapain, ficin and bromelain are reported. The intermediates are dithioacyl enzymes formed during the catalyzed hydrolysis of N-acylglycine thionoester substrates. Interpretation of the resonance Raman spectra allows us to compare, for the first time, the substrate geometries in a series of functioning intermediates from different enzymes. The substrates assume essentially identical conformations for papain, chymopapain and ficin and a similar, but not identical, conformation in the active site of bromelain. Each dithioacyl enzyme population appears to be made up of a single homogeneous conformational state. This state has been characterised in earlier studies of dithioacyl papains. It is designated as conformer B and is characterized by an attractive contact between the substrate's glycinic N atom and the active site cysteine S atom. It is now apparent that conformer B is of general significance in the mechanism of cysteine proteases.     

Citrate synthase stabilizes the enethiolate of acetyldithio coenzyme A

Citrate synthase catalyzes the slow condensation of acetyldithio-CoA [Ac(= S)CoA] with oxalacetate to form thiocitrate [Wlassics, I.D., Stille, C., & Anderson, V.E. (1988) Biochim. Biophys. Acta 952, 269]. During the transient approach to steady state an observable amount of the dithioester absorbance disappears. The amplitude of the decrease in absorbance corresponds to 0.32, 0.03, and 0.02 enzyme equiv at pH 8.3, 7.5, and 6.6, respectively. The difference spectra from before and after the transient exhibit the dithioester lambda max at 306 nm. Acid quenching of a stiochiometric reaction between Ac(= S)CoA and citrate synthase following the transient quantitatively regenerates Ac(= S)CoA, indicating carbon-carbon bond formation had not yet occurred. The apparent first-order rate constant of the transient is independent of Ac(= S)CoA concentration and increases with decreasing pH, being 0.007, 0.016, and 0.04 s-1 at pH 8.3, 7.5, and 6.6, respectively. 2-Fluoroacetyldithio-CoA is a better inhibitor of citrate synthase, Ki = 300 nM, and substrate, Vmax = 2 X 10(-3) s-1, than Ac(= S)CoA. 1H NMR experiments indicate that citrate synthase catalyzes the exchange of the alpha-hydrogens of Ac(= S)CoA with turnover numbers of 0.13 and 0.54 s-1 at pD 7.9 and 7.2, respectively. Analysis of the proton and deuterium decoupled 13C NMR spectra of [2-13C]Ac(= S)CoA that has exchanged 37% of the alpha-hydrogens in the presence of citrate synthase indicates that the relative proportions of CH3, CH2D, CHD2, and CD3 were 0.29, 0.39, 0.25, and 0.07, respectively. This statistical distribution indicates each exchange event is independent. The data indicate that citrate synthase stabilizes the ionized form of Ac(= S)CoA by 5 kcal/mol relative to the un-ionized form, that the ionized dithioester is on the reaction pathway, and that below pH 8.3 the slow carbon-carbon bond forming reaction is responsible for the 10(6) decrease in Vmax caused by substituting sulfur for oxygen in the thioester carbonyl.