Precise structural information for transient enzyme-substrate complexes by a combined x-ray crystallographic-resonance Raman spectroscopic approach

Huber, C. P.; Ozaki, Yoshiharu; Pliura, Diana H.; Storer, Andrew C.; Carey, Paul R.

doi:10.1021/bi00256a012

Cited by 63 publications

(47 citation statements)

References 22 publications

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“…Further evidence that the 1000 cm-' band is found for only A or Cs conformers is that it For personal use only. feature near 1170 cm-' (16), in addition to this feature N-acylalanine dithioesters have a medium intensity peak near 1100 cm-' (Fig. 5).…”

Section: Resonance Rarnan Signatures Of the Different Conformations Omentioning

confidence: 89%

The structure and resonance Raman spectra – structure correlations for methyloxycarbonyl-L-phenylalanyl-L-alanine ethyl dithioester

Varughese¹,

Angus²,

Carey³

et al. 1986

Can. J. Chem.

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The structure of methyloxycarbonyl-L-phenylalanyl-L-alanine ethyl dithioester has been determined by X-ray crystallographic analysis. TheN-C-C-S(thio1) and C-N-C-C(S) torsional angles are 141(1)" and -69(2)", respectively. Consequently, the N and thiono (C=S) sulfur atoms are in close contact and the N...S(thiono) distance is 3.078(5) A. The structure is a perturbed A conformer, which we desknate A', by analogy to a previously characterised A conformer (Huber et al., Biochemistry, 21, 3109 (1982)). The @,angles for the Phe residue are -87(2) and 151(2)", respectively. A Raman spectroscopic study of a single crystal of the LL form and a resonance Rarnan (RR) spectroscopic study of powdered methyloxycarbonyl-L-phenylalanyl-DL-alanine ethyl dithioester establish the RR signature of the dithio moiety for the compound in the A' form. The RR spectrum of the dithioester in CC14 defines the RR signature for the C5 form in solution, while the RR spectrum from a solution in H20/CH3CN provides information on the signatures of the A and B conformers. As for the case of N-benzoyl-DL-alanine dithioester, the A and C5 signatures of the title compound closely resemble those for glycine-based dithioesters but the key marker bands for the B conformer are shifted by =30 cm-' to lower frequency compared to those for a glycine dithioester. 1668 (1986).Faisant appel h la diffraction des rayons-X, on a determink la structure du dithioester Cthylique de la mCthyloxycarbony1 L-phCnylalany1 L-alanjne. [Traduit par la revue]

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Section: Resonance Rarnan Signatures Of the Different Conformations Omentioning

confidence: 89%

The structure and resonance Raman spectra – structure correlations for methyloxycarbonyl-L-phenylalanyl-L-alanine ethyl dithioester

Varughese¹,

Angus²,

Carey³

et al. 1986

Can. J. Chem.

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“…These possibilities include those in binding areas relatively remote from catalytic sites. The crystallographic data for papain [168] permit the construction of models of the acyl-enzyme and tetrahedral intermediates -conclusions supported by the conformations of thionoester substrates bound to papain deduced by resonance Raman spectroscopy combined with crystallography [170,171].…”

Section: Cysteine Proteinasesmentioning

confidence: 91%

Substrate Recognition

Brocklehurst

Gul

Pickersgill

2009

Amino Acids, Peptides and Proteins in Organic Chemistry

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Specific molecular recognition processes by which molecules recognize and discriminate between closely related partners are involved in essentially all aspects of biological function, ranging in complexity from enzyme-inhibitor and enzymesubstrate systems to phenomena such as gene expression, the immune system, and synaptic transmission, and these are important considerations in drug design [1]. This chapter is concerned with general concepts exemplified by a selection of relatively well-defined enzyme-substrate and, by extension, enzyme-inhibitor systems. The original concept of recognition by complementarity in the enzymesubstrate adsorptive (Michaelis) complex was suggested by Emil Fischer in 1894 using his well-known lock-and-key analogy [2]. It is now generally accepted that this has now been superseded by complementarity in the reaction transition state, based on the ideas reported by J.B.S. Haldane [3] in 1930 and elaborated by Linus Pauling [4] in 1946. The catalytic potential of enzymes derives from stabilization in the transition state relative to any stabilization in the Michaelis complex. The latter is anticatalytic in that the free energy expended in stabilizing the adsorptive complex offsets the transition-state stabilization energy (e.g., [5]).Valuable chapters and books on enzyme catalysis and inhibition are available (e.g., [5][6][7][8][9][10][11]) and detailed mechanistic studies on many individual enzymes are described in Sinnot [12]. Macromolecular catalysis includes also catalysis by catalytic antibodies [13] and by synthetic polymers [14], including molecularly imprinted polymers [15]. The contributions of selections of various types of chemical catalysis such as acid-base, nucleophilic, and electrophilic catalysis [5,6,8] are almost always insufficient to account for the high catalytic efficiency of enzymes. A particularly important additional contribution is the entropic advantage that derives from the fact that enzyme catalysis occurs within an enzyme-substrate complex. The complex is essentially a single molecular species and the catalytic act involves one or more unimolecular steps during which there is no loss of translational or rotational entropy

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“…The band appears in the range regarded as normal for vC+ of a N-acylglycine dithioester and may be the signature of any of a number of possible conformers (of which conformer A is but one) possessing a relatively unperturbed dithioester group. Significant rotation about the NHC-C(=S) single bond of conformer B (Huber et al, 1982) will likely attenuate the intramolecular interaction which gives rise to conformer B's atypical RR properties. Thus, with the present data, we can only say that the minor population differs from the major population by a significant (probably >30°) rotation about the NHC-C(=S), and/or possibly the NH-CC(=S), single bond.…”

Section: Discussionmentioning

confidence: 99%

“…The R R spectra, by reference to the spectra of model compounds, allow us to monitor the conformation of the enzyme-bound acyl group in short-lived complexes. The following paper (Huber et al, 1982) deals with joint X-ray and Raman spectroscopic studies on single crystals of model compounds and sets up detailed structure-spectra correlations. These are used with the dithioacyl-enzyme data to provide precise structural information on the acyl-enzyme during catalytic turnover.…”

Section: Resonance R a M A N Study Of Dithioacylpapainsmentioning

confidence: 99%

Vibrational spectra of scissile bonds in enzyme active sites: a resonance Raman study of dithioacylpapains

et al. 1982

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The resonance Raman (RR) spectra of six transient dithioacylpapains have been obtained. For five of these intermediates the dithioacyl group comprises an N-acylglycine dithioester, RC(=O)NHCH,C(=S)S--, while the sixth is a benzoyloxyacetic acid dithioester, C6H5C(=O)OCH2C-(=S)S--. The R R spectra indicate that, for the great majority of molecules in each dithioacylpapain population, the vibrational properties of the dithioester center undergoing catalytic attack are markedly perturbed. By analogy with the R R spectra of models (the corresponding dithioacyl ethyl esters) and by noting the effects of amide hydrogen-deuterium exchange, the source of the perturbation is identified as an intramolecular interaction between the acyl's amide (for the five N-acylglycine dithioester groups) or ester (for the benzoyloxyacetic acid dithioester group) moiety and the dithioester that forms the dithioacylpapain linkage. The R R and ab-I t was demonstrated recently (Storer et al., 1979) that the resonance Raman (RR) spectrum of a dithioacylpapain enables us to monitor the vibrational spectrum of the bonds undergoing catalytic transformation in the enzyme's active site. During catalysis, a transient -C-C(=S)S-linkage is formed in which the substrate and the enzyme contribute the -C-C-(=S)-and -S-moieties, respectively (Lowe & Williams, 1965). The dithioester group has a A , ,near 315 nm, and by use of laser excitation in the 330-nm region, R R bands can be detected that contain contributions from the stretching motions of the C=S bond, which is undergoing nucleophilic attack in the enzyme's active site, and the C-S bond, which is being cleaved during enzymolysis. The R R spectrum of a dithioacyl-enzyme can be used to form a detailed understanding of the structure of the labile group in the active site. However, spectral interpretation was hampered initially by the lack of background spectroscopic information concerning dithioesters. Thus, we instigated a series of infrared, Raman, and RR spectroscopic studies aimed at providing the material required to interpret the R R spectra of dithioacyl-enzymes. The present paper can only be understood by reference to the spectroscopic work and we summarize the important conclusions from these studies as follows:( 1 ) Simple dithioesters of the type CH3C(=S)SCH3 have an intense H -H* electronic transition near 307 nm, and laser excitation within this transition gives rise to intense RR features near 590 and 1195 cm-l. By means of extensive isotopic substitution and a normal coordinate treatment (Teixeira-Dias et al., 1982), these bands are assigned to modes which, for the 590-cm-' feature, have high contributions from stretching motions of the C-C, C=S, and C-S bonds about the C-C-(=S)-S group and, for the 1195-cm-I feature, have a major contribution from C=S stretching coupled to a smaller contribution from C-C stretching. The vibrational spectral regions sorption spectra of the dithioacyl-enzymes are unchanged between pH 4 and 9, but below pH 4 there are marked spectral alterations which o...

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Precise structural information for transient enzyme-substrate complexes by a combined x-ray crystallographic-resonance Raman spectroscopic approach

Cited by 63 publications

References 22 publications

The structure and resonance Raman spectra – structure correlations for methyloxycarbonyl-L-phenylalanyl-L-alanine ethyl dithioester

The structure and resonance Raman spectra – structure correlations for methyloxycarbonyl-L-phenylalanyl-L-alanine ethyl dithioester

Substrate Recognition

Vibrational spectra of scissile bonds in enzyme active sites: a resonance Raman study of dithioacylpapains

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