Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate

Kunugi, Shigeru; Hirohara, Hideo; Nishimura, Eiji; Ise, Norio

doi:10.1016/0003-9861(78)90216-3

Cited by 9 publications

(9 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our investigation indicates, contrary to these proposals, that the intermediate is an artefact, and that steady-state experiments at low pH are consistent solely with the formation and breakdown of the acyl-enzyme. Our observations and interpretations are also in accord with a recent stoppedflow study by Kunugi et al (1978). From the spectral changes and kinetics in the reaction of chymotrypsin with FA-Trp-OMe, they concluded that in the region pH9-5.5 the system is consistent with a simple Michaelis-complex acyl-enzyme pathway, with no other intermediate accumulating.…”

supporting

confidence: 91%

“…(2) Acylation appears as an increase, and deacylation as a decrease, in A320_330 at all pH values between 2.0 and 8.5 (cf. Kunugi et al, 1978). (3) The rates of acylation and deacylation over this pH range are consistent with a single, catalytically significant, ionization of pK 7.0.…”

Section: Discussionmentioning

confidence: 57%

“…The rate of phase 2 was consistent with that of deacylation, based on steady-state experiments and high pH. More recently Kunugi et al (1978) examined the reaction of chymotrypsin with FA-Trp-OMe as a function of pH, by using stopped-flow spectrophotometry. Above pH 5.5, two transients corresponding to phases 1 and 2 were observed; no evidence for a triphasic reaction was detected.…”

Section: Resultsmentioning

confidence: 64%

“…Vol. 181 loyl moiety is particularly well-suited as such a probe for the acyl-amino locus of chymotrypsin substrates (Bernhard et al, 1965), and has previously been so used (Barman & Gutfreund, 1966;Hess et al, 1970;Miller & Bender, 1968;Brot & Bender, 1969;Kunugi et al, 1978). The esters and amides of FA-Trp and FA-Tyr were of especial interest for our cryoenzymological studies, since not only are they specific substrates, but intermediates additional to the Michaelis complex and acyl-enzyme have been reported (Yu & Viswanatha, 1969;Hess et al, 1970;Coletti-Previero et al, 1970).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

Fink¹,

Feldman²,

Zehnder³

1979

Biochemical Journal

View full text Add to dashboard Cite

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347--352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213--217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.

show abstract

supporting

confidence: 91%

Section: Discussionmentioning

confidence: 57%

Section: Resultsmentioning

confidence: 64%

mentioning

confidence: 99%

See 2 more Smart Citations

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

Fink¹,

Feldman²,

Zehnder³

1979

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Data existing in the literature for Z-Xaa-Lys-OMe substrates of trypsin [56], where k,,, follows the same order as in the present Ac-Xaa-Arg-OMe series, as well as Ac-Xaa-Tyr-OMe substrates of chymotrypsin [47] also allow this effect to be recognized. Kunugi et al [48] suggested the enforcement of an unfavourable position of the acylenzyme ester bond relative to His-57 by a tightly binding Pz residue as explanation for the lower k 3 observed for such a chymotrypsin substrate. Increased P3/S3 interactions on substitution of the acetyl group of the dipeptide esters by Z-Pro also lower k,,, (Table 3) (also with tissue kallikrein) in spite of a slight favourable effect on kcat/Km.…”

Section: Effects Of P 2 / S 2 Interactions In Trypsinmentioning

confidence: 99%

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Fiedler¹

1987

European Journal of Biochemistry

View full text Add to dashboard Cite

Kinetic constants for the hydrolysis by porcine tissue /I-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. k,,, and kCa,/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. k,,, for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches k,,, for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. k,,,/K,,, for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. k,,, for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue.In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least Sjof tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well.In sharp contrast to the behaviour of trypsin is the very strong influence of the nature of the P2 residue in tissue-kallikrein-catalyzed reactions. kcal/K,,, varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes k,,,/K, as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 x lo7 M-' s-', suggests rate-limiting binding ( k , ) in the hydrolysis of the best ester substrates. The P2 residue mainly affects K,. The effects of the nature of the P2 residue on the hydrolysis of the dipeptide esters are in accordance with the proposed sandwiching of P2 residues between Tyr-99 and Trp-215 of tissue kallikrein. The pronounced secondary specificity of subsite S2 for bulky, hydrophobic amino acids appears to be a general feature of tissue kallikreins. Remarkably, the two most favourable P2 residues, phenylalanine and leucine, also occur in this position at the two tissue kallikrein cleavage sites of the natural substrate, kininogen. A comparison of the kinetic constants of the kininogen peptides with those for kallidin re...

show abstract

Rapid‐Scanning Stopped‐Flow Spectrophotometry

Brzović

Dunn

1993

Methods of Biochemical Analysis

View full text Add to dashboard Cite

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate

Cited by 9 publications

References 48 publications

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Rapid‐Scanning Stopped‐Flow Spectrophotometry

Contact Info

Product

Resources

About