Chemical Mechanism of the Fructose-6-Phosphate,2-Kinase Reaction from the pH Dependence of Kinetic Parameters of Site-Directed Mutants of Active Site Basic Residues†

Biochemistry ◽  
1997 ◽  
Vol 36 (29) ◽  
pp. 8775-8784 ◽  
Author(s):  
Hiroyuki Mizuguchi ◽  
Paul F. Cook ◽  
Charles A. Hasemann ◽  
Kosaku Uyeda
Keyword(s):  
Kinetic Parameters ◽  
Active Site ◽  
Ph Dependence ◽  
Chemical Mechanism ◽  
Kinase Reaction

scholarly journals Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters

1998 ◽  
Vol 330 (1) ◽  
pp. 311-314 ◽  
Author(s):  
F. RAMÓN ◽  
M. P. CASTILLÓN ◽  
I. DE LA MATA ◽  
C. ACEBAL
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Ph Dependence ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Acid Oxidase ◽  
Protonation State ◽  
Amino Acid Oxidase ◽  
Basic Group ◽  
Rhodotorula Gracilis

The variation of kinetic parameters of D-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. D-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the β-carboxylic group of the inhibitor.

Chemical mechanism of β-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters

Biochimie ◽  
2001 ◽  
Vol 83 (10) ◽  
pp. 961-967 ◽  
Author(s):  
María Gómez ◽  
Pablo Isorna ◽  
Marta Rojo ◽  
Pilar Estrada
Keyword(s):  
Kinetic Parameters ◽  
Trichoderma Reesei ◽  
Ph Dependence ◽  
Chemical Mechanism

Chemical mechanism of penicillin V acylase from Streptomyces lavendulae: pH-dependence of kinetic parameters

2001 ◽  
Vol 16 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Raquel Torres-Guzmán ◽  
Isabel de la Mata ◽  
Jesús Torres-Bacete ◽  
Miguel Arroyo ◽  
Marı́a Pilar Castillón ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Ph Dependence ◽  
Chemical Mechanism ◽  
Penicillin V ◽  
Streptomyces Lavendulae ◽  
Penicillin V Acylase

Chemical mechanism of β-glucosidase from Trichoderma reesei QM 9414. pH-dependence of kinetic parameters

1992 ◽  
Vol 283 (3) ◽  
pp. 679-682 ◽  
Author(s):  
I de la Mata ◽  
P Estrada ◽  
R Macarrón ◽  
J M Dominguez ◽  
M P Castillón ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Trichoderma Reesei ◽  
Carboxy Group ◽  
Ph Dependence ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Histidine Residue ◽  
Activity Temperature ◽  
Beta Glucosidase ◽  
Mechanism Of The Reaction

The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax. and Vmax./Km for p-nitrophenyl beta-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pKi for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.

scholarly journals pH dependence of kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants.

1992 ◽  
Vol 267 (26) ◽  
pp. 18413-18418
Author(s):  
Y Lin ◽  
M Fusek ◽  
X Lin ◽  
J.A. Hartsuck ◽  
F.J. Kezdy ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Kinetic Parameters ◽  
Active Site ◽  
Ph Dependence

scholarly journals Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

1995 ◽  
Vol 308 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
I P Street ◽  
S G Withers
Keyword(s):  
Active Site ◽  
Glycogen Phosphorylase ◽  
Ph Dependence ◽  
Substrate Inhibition ◽  
19F Nmr ◽  
Ionization State ◽  
Nmr Studies ◽  
Substrate Analogues ◽  
Glycogen Phosphorylase B ◽  
Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

1986 ◽  
Vol 64 (4) ◽  
pp. 323-327 ◽  
Author(s):  
Mohammed A. Kashem ◽  
H. Brian Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Ph Dependence ◽  
Transient State ◽  
Nicotinamide Adenine ◽  
Compound I ◽  
Single Ionization ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Kinetics Of

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 °C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I–NADH reaction can be explained in terms of a single ionization of pKa = 4.7 ± 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-H–NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 ± 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II–NADH reaction was observed. Over the pH range of 4–10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 × 105 to5.6 × 102 M−1 s−1 and of HRP-II with NADH varied from 4.4 × 104 to 4.1 M−1 s−1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.

Estimation of kinetic parameters of androgen-synthesizing enzyme activities in superfused Leydig cells from rat testes: Difference between endogenous and exogenous substrates

1984 ◽  
Vol 4 (6) ◽  
pp. 483-488 ◽  
Author(s):  
Nikolaus Kühn-Velten ◽  
Joachim Wolff ◽  
Wolfgang Staib
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Active Site ◽  
Enzyme Activities ◽  
Substrate Concentration ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Endogenous Substrate ◽  
Catalyzed Reactions ◽  
Actual Substrate

Kinetic parameters of 3β-hydroxysteroid dehydrogenase/isomerase, steroid-17α-monooxygenase, and steroid-17,20-lyase activities were estimated under steady-state conditions. Purified Leydig cells from rat testes were superfused with pregnenolone, progesterone, or 17α-hydroxyprogesterone. The Km values for both the monooxygenase- and the lyase-catalyzed reactions were by factors of five to ten higher if analyzed with the exogenously added substrate (0.98 and 0.65 μM, respectively) than if calculated from endogenous substrate derived from a precursor (0.10 and 0.13 μM, respectively). This discrepancy may be explained by different substrate partition between the intra- and extraceIJular spaces and by different substrate concentration at the active site of the respective enzyme, depending on whether the actual substrate is of exogenous or endogenous source.

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