Specificity and stereospecificity of α-chymotrypsin

Ingles, D. W.; Knowles, J. R.

doi:10.1042/bj1040369

Cited by 94 publications

(35 citation statements)

References 22 publications

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“…Similar constraint of a small molecule has been shown to increase a reaction rate by 5 X 104 (75). The ratio of deacylation rates of N-acetyltryptophanyl chymotrypsin compared to its glycyl analogue is 560 (76). This seems to be primarily an entropic effect, due to the orientating effect of the tryptophanyl group.…”

Section: Enzyme Catalysismentioning

confidence: 76%

X-Ray Diffraction Studies of Enzymes

Blow¹,

Steitz²

1970

Annu. Rev. Biochem.

201

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Section: Enzyme Catalysismentioning

confidence: 76%

X-Ray Diffraction Studies of Enzymes

Blow¹,

Steitz²

1970

Annu. Rev. Biochem.

201

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“…The results obtained at pH 8.8 were less unequivocal. A more definite indication of hydrolysis might be expected with an ester having a better leaving group such as thep-nitrophenoxide ion ; however, phenylalanine g-nitrophenyl ester is very unstable in aqueous solution and its hydrolysis is For neutral esters of ~-phenylpropionic acid derivatives, Ingles and Knowles (36,37) found that the -NHof the a-acetamido group was necessary to achieve maximum stereospecificity in the catalytic step subsequent to the binding stage. The 'free' amino acid esters can achieve the same degree of stereospecificity without the interaction of the -NH-(which is now believed to be H bonded to a main-chain carbonyl (38)).…”

Section: Steaesspec~~citymentioning

confidence: 97%

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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“…Enzymes showing stereospecificity toward the aminoacyl residue interact with this residue a t least a t two loci [27]. With peptidyl transferase this condition is met by the binding site for the side chain of the amino acid residue and some other site, such as that for binding the 2'-OH group of ribose as was discussed in a previous paper [13].…”

Section: Products Of the L'ransfer Reactimmentioning

confidence: 99%

Substrate Specificity of Ribosomal Peptidyl Transferase

Rychlík

Černá

Chládek

et al. 1970

European Journal of Biochemistry

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The effect of the nature of the amino acid residue on the acceptor activity of substrates of ribosomal peptidyl transferase was investigated. We tested 2′(3′)‐O‐aminoacyladenosines containing the following amino acid residues: l‐alanine, l‐glutamine, glycine, l‐3‐(1‐benzyl‐4‐imidazolyl)‐alanine, l‐leucine, l‐lysine, l‐methionine, l‐methionine S‐oxide, l‐phenylalanine, d‐phenylalanine, l‐proline, l‐serine and l‐valine as acceptors of the acylaminoacyl residue transferred from peptidyl‐tRNA. The acceptor activity of these compounds was dependent on the nature of the side chain of the amino acid residues bound to adenosine. The acceptor activity was also affected by the nature of the donor tRNA derivative. With l‐acetylphenylalanyl‐tRNA or l‐acetylleucine npetanucleotide fragments derived from l‐acetylleucyl‐tRNA donating the acetylphenylalanyl and the acetylleucyl residue, respectively, a high acceptor activity was shown by the basic 2′(3′)‐O‐l‐lysyladenosine or 2′(3′)‐O‐l‐3‐(1‐benzyl‐4‐imidazolyl)‐alanyladenosine which acted only as weak acceptors of lysine peptides from (Lys)n‐tRNA. The transfer reaction catalyzed by peptidyl transferase was stereospecific with respect to acceptor substrates. The acylaminoacyl residue was transferred from tRNA to 2′(3′)‐O‐l‐phenylalanyladenosine, whereas 2′(3′)‐O‐d‐phenylalanyladenosine, containing a d‐phenylalanine residue, was completely inactive as acceptor.

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Specificity and stereospecificity of α-chymotrypsin

Cited by 94 publications

References 22 publications

X-Ray Diffraction Studies of Enzymes

X-Ray Diffraction Studies of Enzymes

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

Substrate Specificity of Ribosomal Peptidyl Transferase

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