Kinetic salting effect as a promising tool in the investigation of enzyme molecule changes upon reaction: Deacylation of acyl-chymotrypsins

Paberit, Mart

doi:10.1016/0045-2068(90)90045-7

Cited by 5 publications

(2 citation statements)

References 60 publications

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“…enzyme and substrates. At high ionic strengths, the oligopeptides may actually be "salting in" to the enzyme's active site, as has been described for chymotrypsin (Paberit, 1990).…”

Section: Resultsmentioning

confidence: 81%

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by oxygen-18 isotope exchange

Hyland¹,

Tomaszek²,

Roberts³

et al. 1991

Biochemistry

128

135

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The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.

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“…enzyme and substrates. At high ionic strengths, the oligopeptides may actually be "salting in" to the enzyme's active site, as has been described for chymotrypsin (Paberit, 1990).…”

Section: Resultsmentioning

confidence: 81%

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by oxygen-18 isotope exchange

Hyland¹,

Tomaszek²,

Roberts³

et al. 1991

Biochemistry

128

135

View full text Add to dashboard Cite

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“…This observation may indicate dissociation of an ionizable group of the enzyme with pK, value over 11 which has been shown to affect the rates of hydrolysis of various acyl-chymotrypsins [8]. imum' peptide yields at pH 10.5 as the comparable parameters for describing the efficiency of amino acids as acyl acceptors in frozen solutions.…”

Section: Resultsmentioning

confidence: 99%

Peptide synthesis by chymotrypsin in frozen solutions

et al. 1993

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Nucleophilic efficiency of free amino acids in chymotrypsin-catalyzed acyl transfer in ice at -18°C using ethyl esters of N-maleyl-r.-tyrosine and L-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln>Lys>Cit>Ala>Gly>Asn>Arg>Glu>V~>Om>Asp (with no peptide formed with His, Leu, Be and Pro) for N-maleyl-L-tyrosine ethyl ester and Ser>Lys>Om>Arg,Cit>Gln>Thr>Asn>Ala>Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Be and Pro) for L-tyrosine ethyl ester. a-Chymotrypsin; Nucleophile specificity; Frozen solution

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Flavobacterium meningosepticum peptide: N-glycosidase: influence of ionic strength on enzymatic activity

Gosselin

Martin

Murray

et al. 1992

Journal of Biochemical and Biophysical Methods

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Kinetic salting effect as a promising tool in the investigation of enzyme molecule changes upon reaction: Deacylation of acyl-chymotrypsins

Cited by 5 publications

References 60 publications

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by oxygen-18 isotope exchange

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by oxygen-18 isotope exchange

Peptide synthesis by chymotrypsin in frozen solutions

Flavobacterium meningosepticum peptide: N-glycosidase: influence of ionic strength on enzymatic activity

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