Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature.
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ABSTRACT:
SUBMITTER: Mackenzie NE
PROVIDER: S-EPMC1153500 | biostudies-other | 1984 Apr
REPOSITORIES: biostudies-other
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Project description:This study shows for the first time that boric acid catalyses the hydrolysis of peroxyacids, resulting in an approximately 12-fold increase in hydrolysis rate for both peracetic acid (PAA) and 3-chloroperbenzoic acid (MCPBA) when 0.1 M boric acid is present. The maximum rate of hydrolysis occurs at pH 9 and pH 8.4 for PAA and MCPBA respectively. In contrast, carbonate buffer does not enhance the rate of PAA hydrolysis. The reaction was followed by measuring the initial rate of hydrogen peroxide formation using a specific Ti(iv) complexation method. The study of the hydrolysis reaction requires the presence of 2 × 10-5 M each of ethylenediaminetetraacetic acid (EDTA) and ethylenediamine tetramethylene phosphonic acid (EDTMP) in all solutions in order to chelate metal ions across the full pH range (3 to 13) that would otherwise contribute to peroxyacid decomposition. Catalysis of peroxyacid hydrolysis is most likely effected by the triganol boric acid acting as a Lewis acid catalyst, associating with the peroxide leaving group in the transition state to reduce the leaving group basicity. The products of the reaction are the well characterised monoperoxoborate species and the parent carboxylic acid. Analysis of the pH and borate dependence data reveals that in addition to a catalytic pathway involving a single boric acid molecule, there is a significant pathway involving either (a) two boric acid molecules or (b) the polyborate species, B3O3(OH)4 -. Knowledge about catalytic mechanisms for the loss of peroxyacids through hydrolysis is important because they are widely used in reagents in a range of oxidation, bleaching and disinfection applications.
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Project description:The fate of D-[2-13C]glucose 6-phosphate exposed to phosphoglucoisomerase (glucose-6-phosphate isomerase, EC 5.3.1.9) in 2H2O was monitored by 13C-n.m.r. spectroscopy. The generation of the anomers of both D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate followed a single-exponential pattern. The rate constant, which was proportional to the enzyme concentration, was about 14 times higher, however, in the former than in the latter case. The disappearance of D-[2-13C,2-1H]glucose 6-phosphate occurred in a bi-exponential manner, the rate constants for the fast and the slow processes being in fair agreement with those obtained for the generation of D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate respectively. These findings indicate that the process of equilibration of D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate is at least one order of magnitude faster than the intermolecular proton transfer involving the deuterons from the solvent. Such a difference provides strong support to the view that the inverconversion of hexose phosphates in the reaction catalysed by phosphoglucoisomerase proceeds in two distinct steps, the second of which occurs according to two competing modalities with either an intramolecular or an intermolecular proton transfer.
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Project description:The structure of C hordein was studied by a combination of solution and solid-state 13C-n.m.r. spectroscopy. The repetitive primary structure results in simple solution-state n.m.r. spectra, which allow assignment of the majority of the resonances to five major residues. The major resonances, with the exception of the aromatic signals, are also present in the solid-state spectra. The proline residues are in the trans configuration, consistent with an earlier study suggesting a beta-turn-rich structure.
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Project description:To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.
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Project description:The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).
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Project description:A detailed study of the kinetics of the trypsin (EC 3.4.21.4)-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester in cryosolvents at 0 degrees C and below was undertaken. The pH-dependences of kcat, Km, k+2, k+3 and Ks were determined under cryoenzymological conditions and are compared with previous results [Antonini & Ascenzi (1981) J. Biol. Chem. 256, 12449-12455] obtained in fully aqueous media at ambient temperatures. Below pH 5.0 the kinetics, and presumably the mechanism of catalysis, are not significantly perturbed under cryoenzymological conditions. However, it is shown that below pH 5.0 both Km and Ks are decreased under these conditions but that both are increased at pH 6.7 relative to the results obtained in fully aqueous media at ambient temperatures. The effects of the cryoenzymological conditions on the individual catalytic parameters are discussed. The acylation rate constant, k+2, is essentially constant at pH 4.2 and 5.0 but decreases at lower pH values with an apparent pKa of approx. 4.0. In view of the low enthalpy of ionization associated with this pKa it is suggested that this group is the carboxy group of aspartic acid-189, which binds the positively charged lysine side chain of the substrate. The mechanistic implications of the results for the acylation step are discussed. It is also shown that only at low pH values can significant amounts of acylated trypsin be accumulated.
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Project description:The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ?-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M ?-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum ?-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A ?-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.
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Project description:1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives.
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Project description:Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.
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