PubMed Health⌕ Search

Biomedical subjects

R C Wilmouth

Publications and source records attributed to R C Wilmouth.

9 recordsLinked to original sources
PubMed2001

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.

Acylation↗
PubMed2001

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.

Animals↗
PubMed2001

Purification, crystallization and preliminary X-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana.

Anthocyanidin synthase (ANS) from Arabidopsis thaliana is a non-haem iron(II)-dependent dioxygenase reported to catalyse the conversion of leucoanthocyanidins to anthocyanidins. Anthocyanidins are precursors of anthocyanins, which are a major family of pigments in higher plants. ANS was crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 73.2, c = 87.0 A, and diffract to 2.4 A using Cu Kalpha radiation.

Arabidopsis↗
PubMed2000

'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.

beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.

Animals↗
PubMed2000

Analyzing protein functions in four dimensions.

Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1 nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.

Amine Oxidase (Copper-Containing)↗
PubMed1999

Mechanistic insights into the inhibition of serine proteases by monocyclic lactams.

Although originally discovered as inhibitors of pencillin-binding proteins, beta-lactams have more recently found utility as serine protease inhibitors. Indeed through their ability to react irreversibly with nucleophilic serine residues they have proved extraordinarily successful as enzyme inhibitors. Consequently there has been much speculation as to the reason for the general effectiveness of beta-lactams as antibacterials or inhibitors of hydrolytic enzymes. The interaction of analogous beta- and gamma-lactams with a serine protease was investigated. Three series of gamma-lactams based upon monocyclic beta-lactam inhibitors of elastase [Firestone, R. A. et al. (1990) Tetrahedron 46, 2255-2262.] but with an extra methylene group inserted between three of the bonds in the ring were synthesized. Their interaction with porcine pancreatic elastase and their efficacy as inhibitors were evaluated through the use of kinetic, NMR, mass spectrometric, and X-ray crystallographic analyses. The first series, with the methylene group inserted between C-3 and C-4 of the beta-lactam template, were readily hydrolyzed but were inactive or very weakly active as inhibitors. The second series, with the methylene group between C-4 and the nitrogen of the beta-lactam template, were inhibitory and reacted reversibly with PPE to form acyl-enzyme complexes, which were stable with respect to hydrolysis. The third series, with the methylene group inserted between C-2 and C-3, were not hydrolyzed and were not inhibitors consistent with lack of binding to PPE. Comparison of the crystal structure of the acyl-enzyme complex formed between PPE and a second series gamma-lactam and that formed between PPE and a peptide [Wilmouth, R. C., et al. (1997) Nat. Struct. Biol. 4, 456-462.] reveals why the complexes formed with this series were resistant to hydrolysis and suggests ways in which stable acyl-enzyme complexes might be obtained from monocyclic gamma-lactam-based inhibitors.

Animals↗
PubMed1998

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex.

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.

Amino Acid Sequence↗
PubMed1997

Structure of a specific acyl-enzyme complex formed between beta-casomorphin-7 and porcine pancreatic elastase.

Mass spectrometric screening reveals that an unmodified natural heptapeptide--human beta-casomorphin-7, an internal sequence of human beta-casein that possesses opioid-like activity--reacts with porcine pancreatic elastase to form an unusually stable acyl-enzyme complex at low pH. X-ray crystallographic analysis (to 1.9 A resolution) at pH 5 shows continuous electron density linking the C-terminal isoleucine of beta-casomorphin-7 to Ser 195 through an ester bond. The structure reveals a well defined water molecule (Wat 317), equidistant between the carbon of the ester carbonyl and N epsilon 2 of His 57. Deprotonation of Wat 317 will produce a hydroxide ion positioned to attack the ester carbonyl through the favoured Bürgi-Dunitz trajectory.

Animals↗