RESUMEN
SC-51106, a 'minimal-size' diol-based renin inhibitor lacking a P4 residue, has been co-crystallized with human renin and the structure of the complex determined by X-ray crystallography. This study defines the mode of binding of this important class of renin inhibitor, and in conjunction with molecular modeling, has led to the discovery of a new binding site adjacent to S3, which is termed the 'S3aux(iliary)' subsite.
Asunto(s)
Histidina/análogos & derivados , Renina/antagonistas & inhibidores , Renina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Histidina/química , Histidina/metabolismo , Histidina/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Renina/química , Relación Estructura-ActividadRESUMEN
The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-alpha PGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-alpha PGA. The X-ray crystal structure (2.0-A resolution) of MR inactivated by (R)-alpha PGA revealed the presence of a covalent adduct formed by nucleophilic attack of the epsilon-amino group of Lys 166 on the distal carbon on the epoxide ring of (R)-alpha PGA. The proximity of the alpha-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-alpha PGA] was corroborated by the crystal structure (2.1-A resolution) of MR complexed with the substrate analog/competitive inhibitor, (S)-atrolactate [(S)-alpha-methylmandelate]. These results support the proposal that Lys 166 is the polyvalent acid/base responsible for proton transfers on the (S) face of mandelate. In addition, the high-resolution structures also provide insight into the probable interactions of mandelate with the essential Mg2+ and functional groups in the active site.
Asunto(s)
Lisina , Fenilpropionatos/metabolismo , Pseudomonas putida/enzimología , Racemasas y Epimerasas/metabolismo , Alquilación , Cristalografía por Rayos X , Compuestos Epoxi/metabolismo , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Fenilpropionatos/química , Fenilpropionatos/farmacología , Conformación Proteica , Racemasas y Epimerasas/antagonistas & inhibidores , Racemasas y Epimerasas/química , EstereoisomerismoRESUMEN
The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Racemasas y Epimerasas/ultraestructura , Sitios de Unión , Catálisis , Cristalografía , Ácidos Mandélicos/metabolismo , Modelos Moleculares , Conformación Proteica , Pseudomonas/enzimología , Racemasas y Epimerasas/química , Difracción de Rayos XRESUMEN
The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Racemasas y Epimerasas/metabolismo , Secuencia de Bases , Bromuros/metabolismo , Catálisis , Dicroismo Circular , Análisis Mutacional de ADN , Deuterio/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lisina/química , Datos de Secuencia Molecular , Oligonucleótidos/química , Racemasas y Epimerasas/genética , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
The refined structures of two isomorphous pepsin/inhibitor complexes demonstrate that significant conformational changes take place upon ligand binding for a mammalian representative of the aspartic proteinase family. These differences can be attributed mostly to the concerted rigid body movements of two separate clusters of residues relative to a central core. One cluster in the amino domain comprises the flap, the adjacent beta strand (sheet IV) and helices, as well as the interconnecting loops. The other, larger cluster is in the carboxy end and corresponds approximately to the flexible subdomain described previously. Similar conformational changes are proposed to occur in renin and cathepsin D.
Asunto(s)
Pepsina A/química , Animales , Sitios de Unión , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Pepsina A/antagonistas & inhibidores , Conformación Proteica , PorcinosRESUMEN
Mandelate racemase (MR) and muconate lactonizing enzyme (MLE) catalyse separate and mechanistically distinct reactions necessary for the catabolism of aromatic acids by Pseudomonas putida. The X-ray crystal structure of MR, solved at 2.5 A resolution, reveals that the secondary, tertiary and quaternary structures of MR and MLE are remarkably similar; also, MR and MLE are about 26% identical in primary structure. However, MR has no detectable MLE activity and vice versa. Thus, MR and MLE constitute the first example of enzymes that catalyse different reactions, as opposed to mechanistically identical reactions on different substrates, yet possess sufficient structural and sequence identity that they are likely to have evolved from a common ancestor. The discovery that MR and MLE catalyse different reactions but share a common structural framework has broad implications for the natural evolution of enzymes and metabolic pathways, as well as for the rational modification of enzyme activities.
Asunto(s)
Liasas Intramoleculares , Isomerasas/metabolismo , Racemasas y Epimerasas/metabolismo , Sitios de Unión , Evolución Biológica , Catálisis , Cristalización , Isomerasas/química , Estructura Molecular , Pseudomonas/enzimología , Racemasas y Epimerasas/química , Homología de Secuencia de Ácido Nucleico , Difracción de Rayos XRESUMEN
A two-fold (C2) symmetric inhibitor of the protease of human immunodeficiency virus type-1 (HIV-1) has been designed on the basis of the three-dimensional symmetry of the enzyme active site. The symmetric molecule inhibited both protease activity and acute HIV-1 infection in vitro, was at least 10,000-fold more potent against HIV-1 protease than against related enzymes, and appeared to be stable to degradative enzymes. The 2.8 angstrom crystal structure of the inhibitor-enzyme complex demonstrated that the inhibitor binds to the enzyme in a highly symmetric fashion.
Asunto(s)
Endopeptidasas/metabolismo , Productos del Gen pol/metabolismo , VIH-1/enzimología , Inhibidores de Proteasas/farmacología , Alcoholes del Azúcar/farmacología , Valina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Diseño de Fármacos , Proteasa del VIH , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Valina/farmacologíaRESUMEN
We report here the X-ray crystal structure of native subtilisin Carlsberg, solved at 2.5 A resolution by molecular replacement and refined by restrained least squares to a crystallographic residual (Formula see text): of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite 82 amino acid substitutions and one deletion in subtilisin Carlsberg relative to subtilisin BPN', the structures of these enzymes are remarkably similar. We calculate an r.m.s. difference between equivalent alpha-carbon positions in subtilisin Carlsberg and subtilisin BPN' of only 0.55 A. This confirms previous reports of extensive structural homology between these two subtilisins based on X-ray crystal structures of the complex of eglin-c with subtilisin Carlsberg [McPhalen, C.A., Schnebli, H.P. and James, M.N.G. (1985) FEBS Lett., 188, 55; Bode, W., Papamokos, E. and Musil, D. (1987) Eur. J. Biochem., 166, 673-692]. In addition, we find that the native active sites of subtilisins Carlsberg and BPN' are virtually identical. While conservative substitutions at residues 217 and 156 may have subtle effects on the environments of substrate-binding sites S1' and S1 respectively, we find no obvious structural correlate for reports that subtilisins Carlsberg and BPN' differ in their recognition of model substrates. In particular, we find no evidence that the hydrophobic binding pocket S1 in subtilisin Carlsberg is 'deeper', 'narrower' or 'less polar' than the corresponding binding site in subtilisin BPN'.
Asunto(s)
Aminoácidos/análisis , Conformación Proteica , Subtilisinas , Bacillus subtilis/enzimología , Sitios de Unión , Catálisis , Cristalografía , Modelos MolecularesRESUMEN
The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.
Asunto(s)
Isomerasas , Racemasas y Epimerasas , Conformación Proteica , Pseudomonas aeruginosa/enzimología , Difracción de Rayos XRESUMEN
To facilitate large-scale purification and crystallographic study, we have subcloned the gene for the alanine racemase of Bacillus stearothermophilus from pICR401 (Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., and Soda, K. (1986) Biochemistry 25, 3268-3274) and overproduced the enzyme in Escherichia coli W3110 lacIq using the tac promoter of PKK223-3. This system yields alanine racemase as 6% of the bacterial cytosolic protein. Purification by a modification of the procedure of Inagake et al. yielded 75 mg of homogeneous alanine racemase from 30 g of cells (wet weight). Large, well-formed crystals of alanine racemase have been grown from polyethylene glycol 8000 using vapor diffusion. These crystals have unit cell dimensions a = 85.3 A, b = 110.0 A, and c = 89.9 A. The crystals belong to space group P2(1), with beta fortuitously equal to 90 degrees within experimental error; however, they are frequently twinned by second order pseudomerohedry with twin fraction (the ratio of the volume of the smaller twin domain to the total volume of the crystal) ranging from about 0 to 0.5. Fortunately, for crystals with low twin fraction, computational methods have been developed for the analysis and correction of simple twinning (Fisher, R. G., and Sweet, R. M. (1980) Acta Crystallogr. A36, 755-760). The crystals contain two alpha 2 dimers of alanine racemase in the asymmetric unit. We have identified several potentially useful heavy atom derivatives in low resolution screening experiments and are proceeding with high resolution data collection.