A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

Elmore, D. T.; Smyth, J. J.

doi:10.1042/bj1070103

Cited by 48 publications

(19 citation statements)

References 16 publications

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“…It is also catalyzed by carbon dioxide (35). As with interesting that Elmore and Smyth (34) could the N-acetyl analogues the D-isomers inhibit the not use N-methyl-N-tosyl-L-phenylalanine 3-hydrolysis of the L-esters. In the case of D-PNBE pyridyl ester as an active site titrant because the the inhibition was shown to be competitive.…”

Section: Specijicitymentioning

confidence: 98%

The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group

Je¹,

Nl²

1970

Can. J. Biochem.

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The interaction of a-chyrnotrypsin with phenylalanine derivatives containing a free a-amino group. Can. J. Biochem. 48,1058Biochem. 48, -1065Biochem. 48, (1970.The action sf or-chymotrypsin on L-and D-phenylalanine ethyl esters (PEE), L-and D-phenylalanine p-nitrobenzyl esters (PNBE), L-phenylalmine methyl and isopropyl esters, and N-methyl-L-phenylalanine methyl ester has been studied using a pH-stat. The D-esters were not hydrolyzed but acted as competitive inhibitors of the hydrolysis of the L-isomers. The N-methyl ester was very slowly hydrolyzed due to its Iow kc,,. For L-PEE (pK 7.23) and L-PNBE (pK 6.931, the activity of a-chymotrypsin is displaced to a more acid region relative to that for the N-acyl amino acid esters. The Km increases sharply below pH 6.5 while the kc,, and k,,,/Km show maxima at pH 6 and 5.6, respectively. On the acid side kc,, is controlled by a basic group of pK 4.86 for L-PNBE and pK 5.1 for L-PEE, and kc,,/Km by a basic group s f p r 6.4 for L-PNBE and pK6.6 for L-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the cata1ytlcall.y active entities of the enzyme-substrate complex and acyl enzyme, the a-amino group of the substrate IS protsnated, (iii) the pK of the basic group on the acyl enzyme is considerably lowered by the presence t of the a-NH3 of the substrate, and (iv) the increase in Km and Kf below pH 6.8 is due to the development of unfavorable charge interactions. Introductionan a-acyl amino group by an amino group leads Prior to the work of Petitc1el-c and Benoiton to a partial loss in stereospecificity4 (7)-The (1) on ~-t~r~~i~~ ethyl ester, there had been no present paper describes some steady-state kinetic study of the p H dependence of the parameters studies of the interaction of i*-chymotry~sin k,,,3 and K, for the r*-chymotrypsin-catalyzed with several derivatives of phenylalanine conhydrolysis of free amino acid esters. It had been taining free a-amino groups. reported that the pH optima for the hydrolysis of L-tyrosine ethyl ester, L-tyrosine hydrazide, and L-tyrosine kydroxamide were 6.5, 7.0, and 6.95, respectively (2-41, and kc,, and .Km values had been derived for the latter. Petitclerc (5) has shown that in contrast to the results obtained for N-acyl amino acid esters (ti), the hydrolysis of L-tyrosine ethyl ester is characterized by a strong pH dependence of Km below pH 7 and by a sharp optimum in kc,, at pH 6.1. Moreover, he was unable to detect any hydro~ysis of the D-enantiomer although Hein and Niemann have stated that besides leading to a marked decrease in the rate of hydrolysis, replacement of

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Section: Specijicitymentioning

confidence: 98%

The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group

Je¹,

Nl²

1970

Can. J. Biochem.

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“…Only a 5 impurity of molecular weight 40 000 was detected which was enzymatically inactive. The molar absorption coefficient of the enzyme = 5.ix104 M -1 cm-' was determined by active-site titration with 4-nitrophenyl-N'-acetyI-N1-benzylcarbazate according to Elmore and Smyth [17]. Proflavine was purchased from Fluka (Fuchs, Switzerland) and 4-nitrophenyl-NL-acetyl-N1-benzylcarbazate from Nutritional Biochemicals Corp. (Cleveland, Ohio).…”

Section: Methodsmentioning

confidence: 99%

Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Quast¹,

Engel²,

Steffen³

et al. 1978

European Journal of Biochemistry

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Modified trypsin kallikrein inhibitor (I*), with the reactive‐site peptide bond Lys‐15–Ala‐16 split, reacts with α‐chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C: E + I*⇌ X → C. Formation of X constitutes a fast pre‐equilibrium (equilibrium constant Kx= 7 × 10−5 M. association rate constant kx= 4 × 103 M−1 s−1) to the slow reaction X → C (rate constant kc= 2 × 10−3 s−1), all values at pH 7.5. No intermediate X is observed when α‐chymotrypsin reacts with I*‐OMe in which the carboxyl group of Lys‐15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*‐OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with β‐trypsin and I* or I*‐OMe.

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“…Incubation of the treated enzyme with dithiothreitol a t 5 mM fully restored the original activity. If one assumes that there is one mole of reactive sulphhydryl per equivalent of competent enzyme active site, that essentially all such sites are kinetically equivalent and that the reactive sulphhydryl titre equals the operational normality [21,27,28] of the enzyme, then the absolute activity of the active enzyme would be 865/(36.7 x lo6) = 2.3 x lo7 units per equivalent, independent of the estimate of molecular weight. Then, conversely, a rough estimate of the operational normality of an enzyme preparation of undetermined sulphhydryl titre can be obtained as the ratio (enzymic activity in mU/m1)/2.3 x 107.…”

Section: 4'-dithiodi~yridinementioning

confidence: 99%

Anionic Proteinase from Actinidia chinensis. Preparation and Properties of the Crystalline Enzyme

McDowall

1970

Eur J Biochem

100

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1. A method is described for the preparation from Actinidia chinensis (Chinese gooseberry) fruit of a crystalline proteinase which catalyzes the hydrolysis of N2-benzoyl-L-arginine ethyl ester, N2-p-toluene-sulphonyl-L-glutamine p-nitrophenyl ester (used in the routine assay) and a t least 15 o f the peptide bonds of gelatin. The synthesis of the tosyl-glutamine p-nitrophenyl ester is described. The enzyme resembles papain in its action against benzoyl-L-arginine ethyl ester, having a broad pH-optimum from pH 5-7, with K,(app) of 89 mM and keat of about 2.6 sec-l at pH 5.6, 25" in 0.3 M KCI.2. The enzymic activity elutes from Sephadex G-50 as a protein of molecular weight 12800 f 700, and is partially resolved from inactive protein of apparent molecular weight 15400 f 800. The preparation has minimum solubility near pH 3.1 and migrates a t both pH 8.3 and 5.0 on polyacrylamide gel electrophoresis as two closely-spaced anionic bands. These two electrophoretic components are partially resolved by chromatography on DEAE-cellulose and both are enzymically active against tosyl-L-glutamine p-nitrophenyl ester.3. The inhibitions of enzymic activity by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiodipyridine, both reversible by 1,4-dithiothreitol, show that enzymic activity is dependent on an intact sulphhydryl group assaying about 0.23 moles per 12800 g protein. The enzyme is rapidly inactivated by iodoacetate and thereafter gives a low sulphhydryl assay with dithiodipyridine. Conversely, pretreatment with DTNB gives substantial protection against iodoacetate.The proteinase activity in extracts of New Zealand commercial strains of the ripe fruit of the Chinese Gooseberry, Actinidia chinensis has been characterized by Arcus [1] by its activity against gelatin (optimum p H 4 ) and haemoglobin, and was shown to be enhanced by cysteine, Na,S, KCN and EDTA. It is of interest to isolate the enzyme(s) responsible for this activity because of possible structural homologies with the sulphhydryl plant proteinases papain, ficin, bromelin, chymopapain B and pinguanain [2,3], especially now that the tertiary structure of crystalline papain has been elucidated [4].This report describes the preparation of a crystalline anionic proteinase from A. chinensis, some physical and enzymic properties, and further evidence for the requirement of a sulphhydryl group for activity.Unusual Abbreviations. DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); CNps-proteinase, 3-carboxy-4-nitrophenylsulphenyl-proteinase; BzArgOEt, N2-benzoyl-L-arginine ethyl ester hydrochloride ; TosGlnONp, N2-p-toluenesulphonyl-L-glutamine p-nitrophenyl ester; Pr,P-trypsin, diisopropylphosphoryl-trypsin ; V , and V,, void volume and elution volume.

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A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

Cited by 48 publications

References 16 publications

The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group

The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group

Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Anionic Proteinase from Actinidia chinensis. Preparation and Properties of the Crystalline Enzyme

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