Kinetics of Hydrolysis of Dansyl Peptide Substrates by Thermolysin: Analysis of Fluorescence Changes and Determination of Steady-State Kinetic Parameters

Biochemistry ◽  
1994 ◽  
Vol 33 (21) ◽  
pp. 6508-6515 ◽  
Author(s):  
Jenny Jie Yang ◽  
Harold E. Van Wart
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Peptide Substrates ◽  
Kinetics Of Hydrolysis ◽  
Steady State Kinetic ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

2007 ◽  
Vol 365 (2) ◽  
pp. 165-173 ◽  
Author(s):  
Janne Jänis ◽  
Petri Pulkkinen ◽  
Juha Rouvinen ◽  
Pirjo Vainiotalo
Keyword(s):  
Mass Spectrometry ◽  
Steady State ◽  
Kinetic Parameters ◽  
Steady State Kinetic ◽  
Hydrolysis Of ◽  
State Kinetic

The Steady State Kinetics of Plasmin and Trypsin Catalysed Hydrolysis of a Number of Tripeptideanilides

1979 ◽  
Author(s):  
U. Christensen ◽  
H-H. Ipsen
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

The steady state kinetic parameters of plasmin and trypsin catalysed hydrolysis of Bz-L-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA in the pH-range 6-9 are presented. Ionization of catalytically essential enzymic groups accounts satisfactorily for the pH-dependencies of the kinetic parameters for plas-rain and trypsin reactions with Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA. The protonation of the α-amino group of L-Phe-Val-Arg-pNA and D-Phe-Val-Arg-pNA (pK=7.0) show some additional effect. The values of the catalytic constants for plasmin and trypsin reactions with these p-nitroanilides are alike those normally found for specific ester substrates, indicating that the deacylation steps are rate determining.

scholarly journals Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS

2003 ◽  
Vol 374 (2) ◽  
pp. 369-380 ◽  
Author(s):  
Omid HEKMAT ◽  
Ken TOKUYASU ◽  
Stephen G. WITHERS
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Intrinsic Rate ◽  
Degree Of Polymerization ◽  
Colletotrichum Lindemuthianum ◽  
Chitin Deacetylase ◽  
Steady State Kinetic ◽  
Intrinsic Rate Constant ◽  
Hydrolysis Of ◽  
State Kinetic

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed Km and kcat/Km on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (kcat, 7 s−1) is independent of n and represents the intrinsic rate constant (kint) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed kcat/Km values (A−2, −11.0; A−1, −1.5; A0, −7.7; A+1, −12.5 kJ·mol−1). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [ΔΔG(N-acetyl)=3.3, 0, 4.0 and 0 kJ·mol−1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.

Steady-state kinetics of hydrolysis of dansyl-peptide substrates by leucine aminopeptidase

Biochemistry ◽  
1988 ◽  
Vol 27 (14) ◽  
pp. 5062-5068 ◽  
Author(s):  
Wann Yin Lin ◽  
Spencer H. Lin ◽  
Harold E. Van Wart
Keyword(s):  
Steady State ◽  
Leucine Aminopeptidase ◽  
Peptide Substrates ◽  
Kinetics Of Hydrolysis ◽  
Steady State Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of

scholarly journals Interpretation of the Kinetics of Consecutive Enzyme-catalysed Reactions: Studies on the Arginase-Ornithine Carbamoyltransferase System

1982 ◽  
Vol 35 (2) ◽  
pp. 137 ◽  
Author(s):  
RD Teasdale ◽  
PD Jeffrey ◽  
PW Kuchel ◽  
LW Nichol
Keyword(s):  
Steady State ◽  
Ornithine Carbamoyltransferase ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Kinetic Mechanisms ◽  
Heterogeneous Association ◽  
Kinetics Of ◽  
State Kinetic ◽  
Enzymic Assay

A method that permits the use of measurements on the concentration of the intermediate in a coupled enzymic assay in determining the presence or absence of an interaction between the enzymes is presented. The method is shown to be closely analogous to a previously formulated procedure involving the determination of the rate of production of the final product of such a sequence and is shown to be applicable regardless of the complexity of the operative kinetic mechanisms, provided it may be assumed that all enzyme-substrate complexes are in the steady-state. Kinetic results obtained with the arginase--ornithine carbamoyltransferase couple, in which the intermediate ornithine is monitored, are examined in these terms to conclude that no heterogeneous association is operative between the enzymes.

scholarly journals Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin.

1983 ◽  
Vol 258 (15) ◽  
pp. 9276-9282 ◽  
Author(s):  
D L Higgins ◽  
S D Lewis ◽  
J A Shafer
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Human Fibrinogen ◽  
Steady State Kinetic ◽  
State Kinetic
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