An Electrophilic Mechanism in the Chymotrypsin-Catalyzed Hydrolysis of Anilide Substrates<sup>1</sup>

Inagami, Tadashi; York, Sheldon S.; Patchornik, A.

doi:10.1021/ja01079a027

Cited by 39 publications

(21 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was shown that the reaction went through a covalent acyl-enzyme intermediate (145) and the deuterium isotope effect indicated that a proton transfer step was rate limiting in some circumstances (146 ). On this basis, and from preliminary crystallographic results (128 ), a mechanism was pro posed in which, for certain types of substrates, a proton was transferred from the active serine to a nearby histidine residue, His 57, in a pretransi tion state equilibrium (147).…”

Section: Chymotrypsinmentioning

confidence: 99%

X-Ray Diffraction Studies of Enzymes

Blow¹,

Steitz²

1970

Annu. Rev. Biochem.

201

View full text Add to dashboard Cite

Section: Chymotrypsinmentioning

confidence: 99%

X-Ray Diffraction Studies of Enzymes

Blow¹,

Steitz²

1970

Annu. Rev. Biochem.

201

View full text Add to dashboard Cite

“…It would work against k3 because that step requires the transfer of a proton to the leaving group. However, kCat/K,,,, for the hydrolysis of Ac-Tyr-Nan is 72 M-' s-' [34] compared to 18 M-' s-' for the less electronegative acetyl-Ltyrosine p-methoxyanilide [35]. It appears then that the enzyme has a mechanism to facilitate proton transfer despite a barrier posed by a highly electronegative leaving group.…”

Section: Discussionmentioning

confidence: 97%

Allosteric Activation of the Hydrolysis of Specific Substrates by Chymotrypsin

et al. 1976

View full text Add to dashboard Cite

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...

show abstract

“…For example, although a good correlation exists between the acylation rates (keat) of chymotrypsin by a series of ring substituted N-acetyl-tyrosine anilides and o-, the rate constant for the corresponding p-nitroanilide is fasted than predicted by a factor of over 100 [3]. In addition, acylation of chymotrypsin by nitro-substituted N-benzoyltyrosine anilides shows a rate enhancement as electron-withdrawal increases [5], whereas for other substituents the rate is increased by electron-donating groups [2][3][4].…”

Section: Introductionmentioning

confidence: 94%

“…They have been used as substrates for trypsin [1 ], chymotrypsin [2][3][4][5], elastase [6,7], aminopeptidase M [8], papain [1,9] and subtilisn [10]. It is unclear, however, whether p-nitroanilides are normal substrates for these enzymes.…”

Section: Introductionmentioning

confidence: 99%