Allosteric Activation of the Hydrolysis of Specific Substrates by Chymotrypsin

Abstract

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolyis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[a-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-Q-Bzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing k,,, without altering K,. The hydrolysis of acetyl-Ltyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reactivation in the presence of NH20H was also accelerated, but in the absence of added nucleophile (ie. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[or-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in k,,, of hydrolysis; K , was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limiting, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule. In a preliminary communication [l] we reported acceleration of the chymotryptic hydrolysis of certainAbbreviations. Glt-Phe-Nan, glutaryl-L-phenylalanine p-nitroanilide ; Ac-Tyr-Nan, a-N-acetyl-DL-tyrosine p-nitroanilide; Z-Tyr-NHOH, carbobenzoxy-L-tyrosine hydroxamide; Ac-Tyr-An,, a-N-acetyl-L-tyrosine anilide ; Q-Me, bis[a-(trimethy1ammonium)methyllazobenzene dibromide; Q-Bzl, bis[a-(benzyldimethylammonium)methyl]azobenzene dibromide; Q-Nbzl, bis[a-(p-nitrobenzyldimethylammonium)methyl]azobenzene dibromide; Q-BrMeBzl, 2,2' -bis[a-(0 -bromomethylbenzyldimethylammonium)methyl]azobenzene dibromide; K-Bzl, 2,2'-[a-(benzy1dimethylammonium)-methyl]diketobenzene dibromide; Ac-Tyr-OEt, a-N-acetyl-L-tyrosine ethyl ester; Ac-Gly-OEt, a-A'-acetyl-glycine ethyl ester; Bz-GlyOEt, a-N-benzoyl-glycine ethyl ester; PhZNCF and Ph,NCchymotrypsin, diphenylcarbamyl chloride, fluoride and chymotrypsin, respectively; Ac-ONp, p-nitrophenylacetate ; Bz-Arg-Nan, a-Nbenzoyl-DL-arginine p-nitroanilide.Enzyme. a-Chymotrypsin or chymotrypsinogen A (EC 3.4.21.1).specific substrates by a number of compounds having the general structure :QMe: R=CH3 cis and rrans isomers Q-BrMeBzl: R=CH2Acceleration of substrate hydrolysis (for example, Glt-Phe-Nan) occurred as a result of the binding of the activator molecule to a site...

SIRT1 Activation by Small Molecules

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Stimulus‐Responsive Regulation of Enzyme Activity for One‐Step and Multi‐Step Syntheses

“…α‐amylase;– β‐amylase; urease; α‐chymotrypsin; β‐glucosidase; aldolase; papain; horseradish peroxidase…”

“…The random incorporation of photo‐switchable residues, which was mostly performed at the beginning, is therefore no longer that common. Changes in enzyme activity through the random incorporation of photo‐switchable moieties were achieved in earlier days, for example, for α‐amylase,– β‐amylase, urease, α‐chymotrypsin, β‐glucosidase, aldolase and papain by incorporating spiropyran– or azobenzene moieties.…”

Allosteric Site of Serine Proteinases: Localization, Functional Role and Manifestations in Vitro

“…N-dansyl-Larginine also exhibits the properties of allosteric trypsin effector due to the binding both to the active site of the enzyme and outside it [12]. Salts of trialkylammonium derivatives of azobenzene accelerate α-chymotrypsin-mediated hydrolysis of Nacetyl-amino acid anilides [13]. Hydrolysis by α-chymotrypsin of a nonspecific substrate -acetyl-p-nitrophenyl ether -is accelerated by a large number of hydrophobic compounds [14,15].…”

“…Hydrolysis by α-chymotrypsin of a nonspecific substrate -acetyl-p-nitrophenyl ether -is accelerated by a large number of hydrophobic compounds [14,15]. The activation region of chymotrypsin is thought to be similar to the binding site, although more polar, and located somewhere on the surface of the enzyme molecule [13]. Data on reactions in such kind of systems suggest that a protein capable of simultaneously interacting with both enzyme's binding site and allosteric one will be subject to much faster hydrolysis than in the case of a single 190 SECTION XI.…”