Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence- and stereospecificity of the S1- and S' i-subsites (i = 1-3) of bovine alpha-chymotrypsin and trypsin, proteinase K and penicillin amidase from E. coli and A. viscosus has been determined by hydrolysis and kinetically controlled peptide synthesis using different substrates. The data are compared with results for other serine proteases and the thiol protease papain. The stereospecificities differ by orders of magnitude, decreased when the enzyme was immobilized and were influenced when organic solvent molecules were bound to the enzyme.
 ...
PMID:Stereo- and sequence specificity of serine proteases in peptide synthesis. 182 58

Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F-inactivated chymotrypsin [Gold, A.M. & Fahrney, D. (1964) Biochemistry 3, 783-791]. Incubation of the PhMeSO2F-inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol-enzyme during incubation with 10 mM 6-aminopenicillanic acid. 4-Pyridyl-ethylcysteine was released by acid hydrolysis after reaction of the thiol-protein with 4-vinylpyridine. The rates of reaction of thiol-penicillin acylase with iodoacetic acid and 2,2'-dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol-enzyme with iodo[2-3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the beta-chain NH2-terminal residue, indicating conversion of Ser290 to S-carboxymethyl-cysteine. Near-ultraviolet CD spectra showed the conformation of thiol-penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.
 ...
PMID:Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity. 184 24

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.
 ...
PMID:Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability. 397 68

Immobilization of enzymes (penicillin amidase and alpha-chymotrypsin) in water-soluble nonstoichiometric polyeloctrolyte complexes (PEC) formed by poly(4-vinyl-N-ethylpyridinium bromide) (polycation) and polymethacrylic acid (polyanion) was carried out. Particles of these PEC consist of a nucleus formed by sequences of salt bonds between the units of oppositely charged polyelectrolytes and the hydrophylic shell formed by ionized groups of polyanions which is in excess in PEC. Such a structure of PEC particles results in a cooperative phase transitions of these systems at slight variations of pH and ionic strength. The work demonstrates phase diagrams of PEC solutions. The values of pH and ionic strength at which phase transitions in solutions of different PEC occur were elucidated. The decrease of pH value from 6.1 to 5.7 leads to reversible phase transition followed by a saltatory increase of Km for immobilized penicillin amidase by 5-10 fold depending on substrate used. The phase transition induced by ionic strength increase up to 0,27 M NaCl doesn't change significantly the Km-value of enzymic reaction. The phenomenon observed can be accounted for by the different structure of PEC particles. The catalytic properties of immobilized chymotrypsin were shown to depend on the loci of enzyme attachment. If the enzyme is bound to polyanion, neither conformational changes of the matrix nor phase transition in solution influence its accessibility for the protein inhibitor, but rather change the binding constant. If the enzyme is attached to polycation, i.e. included in the polycomplex nucleus, two fractions of enzymes accessible and inaccessible for protein inhibitor appear.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:[Enzymes incorporated in polyelectrolyte complexes. Effect of matrix conformational changes and phase transitions in solutions on catalytic properties]. 635 18

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.
 ...
PMID:Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product. 1862 46