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K Brocklehurst

Publications and source records attributed to K Brocklehurst.

At least 37 records · Page 2Linked to original sources
PubMed1996

Comparative studies on the 5-aminolaevulinic acid dehydratases from Pisum sativum, Escherichia coli and Saccharomyces cerevisiae.

5-Aminolaevulinic acid dehydratase (ALAD) is an essential enzyme in most organisms, catalysing an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr-type condensation reaction of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole porphobilinogen. ALADs can be conveniently separated into two main groups: those requiring Zn2+ for activity (typified here by the enzymes from Escherichia coli and Saccharomyces cerevisiae, yeast) and those requiring Mg2+ (represented here by the enzyme from Pisum sativum, pea). Here we describe a detailed comparison of these two metal-dependent systems. Kinetically influential ionizations were identified by using pH-dependent kinetics. Groups with pKa values of approx. 7 and 10 (assigned to cysteine and lysine residues) were detected in the free enzyme and enzyme-substrate states of all three enzymes, and a further ionizable group with a pKa of approx. 8.5 (assigned to histidine) was found to be additionally important to the yeast enzyme. The importance of these residues was confirmed by using protein modifying reagents. Shifts in the pKa values of the pea and E. coli enzymes consequent on E-S complex formation suggest a change to a less hydrophobic micro-environment when substrate binds. Studies with inhibitors revealed that the three enzymes exhibit differential susceptibilities and, in the case of succinylacetone, this is reflected in Ki values that vary by three orders of magnitude. In addition, the crystallization of the yeast ALAD is described, raising the possibility of an X-ray-derived three-dimensional structure of this enzyme.

Amino Acid Sequence↗
PubMed1996

Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-beta-trypsin complex.

1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The K1, value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme. Computer modelling of the beta-trypsin-E64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

Amino Acid Sequence↗
PubMed1996

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

2,2'-Dipyridyl↗
PubMed1996

The house dust mite allergen Der p1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma.

Der p1, a cysteine proteinase derived from the house dust mite (HDM) Dermatophagoides pteronyssinus, is a major component of the allergic immune response in HDM atopic individuals. Recent evidence suggests that cysteine proteinase activity is important in the disease process as it increases the permeability of the allergen in the respiratory tract and disrupts the regulation of IgE synthesis. Der p1 is found in high concentrations in the faecal pellets of mites which are aerosolised and inhaled via the respiratory tract. The serine proteinase inhibitor, alpha 1-antitrypsin, protects the lower respiratory tract against damage by proteinases released in the lung during inflammation. Der p1 catalytically inactivates alpha 1-antitrypsin by a thiol-dependent mechanism involving specific cleavage of the reactive centre loop and we propose that this mechanism may be important in the pathogenesis of asthma.

Allergens↗
PubMed1995

Evidence for the importance of weakly bound water for matrix metalloproteinase activity.

The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated. In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates. Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme. The former reflects the hydrophobic nature of the enzyme-substrate interaction and the effect can be corrected for by using the solvent to water partition coefficient of the substrate in the mixed solvent systems. The catalyzed hydrolysis of substrate, corrected for the effect of hydrophobic partitioning, was demonstrated to be sixth order in water for gelatinase A and third order in water for stromelysin 1. Variation in water concentration did not produce saturation even at concentrations close to 55.5 M. The results indicate that weakly bound water molecules are essential to mediate the interaction between substrate and enzyme. The sensitivity of these enzymes to water concentration could be an important mechanism for regulating catalytic activity in vivo.

Amino Acid Sequence↗
PubMed1995

The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin.

1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2'-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric 'cap' resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.

Binding Sites↗
PubMed1995

The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full-length enzymes: a possible role for the C-terminal domain.

Domain deletion mutants of the matrix metalloproteinases consisting of the catalytic domain only contain two zinc atoms per molecule. One is essential for catalysis, while the other may fulfil a structural role. We have analysed the zinc contents of both the full-length and the truncated mutants of prostromelysin-1 and progelatinase A and report that the second zinc atom is not present in the full-length form of the proenzymes. Thus it seems likely that the role proposed for this zinc atom in maintaining the structure of the enzyme catalytic domain is performed by the C-terminal domain in the full-length enzyme.

Amino Acid Sequence↗
PubMed1995

Catalytic site targeted mutagenesis of the alpha-gingivain gene of Porphyromonas gingivalis using Tn-4351 to generate isogenic mutants.

The extracellular proteinases of the anaerobe Porphyromonas gingivalis, are implicated in the destruction of host defence mechanisms in periodontitis. We have previously purified one of these enzymes, alpha-gingivain, and established that it belongs to the cysteine proteinase family of enzymes. In the present study, transposon Tn4351 was used to alter the open reading frame encoding a region that includes the catalytic site of alpha-gingivain by targeted mutagenesis. Escherichia coli HB101 which harbours R751 was used to introduce the transposon into P. gingivalis ATCC 33277 by conjugal transfer. E. coli was transformed using the altered plasmid with a Cla I site insertion of a sequence common to the catalytic site histidine or cysteine of many cysteine proteinases. The frequency of the transconjugation was 4.5 x 10(5) while the recipient viable counts comprised 60% of the original P. gingivalis. The result of this targeted mutagenesis was inactivation of gingivains such that some colonies on skimmed-milk agar plates showed no clear surrounding zones of hydrolysis and their normal catalytic activity towards L-BAPNA was destroyed.

Journal Article↗
PubMed1994

Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2'-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4'-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗