pH Dependence and competitive product inhibition of the carboxypeptidase A catalyzed hydrolysis of O-(trans-cinnamoyl) L-.beta.-phenyllactate

1969 ◽  
Vol 91 (2) ◽  
pp. 485-491 ◽  
Author(s):  
Philip L. Hall ◽  
B. L. Kaiser ◽  
Emil T. Kaiser
Keyword(s):  
Ph Dependence ◽  
Product Inhibition ◽  
Carboxypeptidase A ◽  
Hydrolysis Of ◽  
Competitive Product

The pH dependence of the hydrolysis of hippuric acid esters by carboxypeptidase A

Biochemistry ◽  
1976 ◽  
Vol 15 (15) ◽  
pp. 3237-3244 ◽  
Author(s):  
John W. Bunting ◽  
Samuel S. T. Chu
Keyword(s):  
Hippuric Acid ◽  
Ph Dependence ◽  
Carboxypeptidase A ◽  
Hydrolysis Of ◽  
Acid Esters

Kinetics of the hydrolysis of cellulose by β-1,4-glucan cellobiohydrolase of Trichoderma viride

1977 ◽  
Vol 55 (6) ◽  
pp. 644-650 ◽  
Author(s):  
R. James Maguire
Keyword(s):  
Time Course ◽  
Trichoderma Viride ◽  
Kinetic Equations ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Cellulolytic Enzyme ◽  
Hydrolysis Of Cellulose ◽  
Ph Curve ◽  
Hydrolysis Of ◽  
Competitive Product

The cellulolytic enzyme β-1,4-glucan cellobiohydrolase (CBH) has been isolated from the crude mixture of cellulase enzymes of Trichoderma viride by gel filtration and ion-exchange methods, and some aspects of its kinetic behaviour have been examined. Studies of the initial rates of the CBH-catalyzed production of cellobiose from fibrous α-cellulose show that (i) the dissociation constant for cellobiose competitive product inhibition of the reaction is Ki = (1.13 ± 0.37) × 10−3 M, (ii) the adsorption of CBH on fibrous α-cellulose and its subsequent reaction conform to kinetic equations developed in conjunction with the Langmuir adsorption isotherm, (iii) the rate–pH curve has a maximum at pH 5.2 and decreases at higher and lower pH values, exhibiting enzyme pK values of 3.8 and 6.5, and (iv) the energy of activation of the overall reaction between 5 and 60 °C is 5.3 ± 0.3 kcal mol−1 at pH 5.2. Studies of the time course of the reaction over extended periods of time up to 40% hydrolysis of the cellulose show that (v) the data fit better to a competitive product inhibition model than to models of anticompetitive product inhibition or noncompetitive product inhibition.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Substituent Effects in the Carboxypeptidase A Catalyzed Hydrolysis of Substituted L,.beta.-Phenyllactate Esters

Biochemistry ◽  
1994 ◽  
Vol 33 (49) ◽  
pp. 14750-14757 ◽  
Author(s):  
Alan Osumi ◽  
Abdulkader Rahmo ◽  
Stephen W. King ◽  
Theodore J. Przystas ◽  
Thomas H. Fife
Keyword(s):  
Substituent Effects ◽  
Carboxypeptidase A ◽  
Hydrolysis Of

pH Effects in trypsin catalysis

1969 ◽  
Vol 47 (21) ◽  
pp. 4021-4029 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Complex Formation ◽  
Ph Dependence ◽  
Specific Rotation ◽  
Ph Effects ◽  
Ph Values ◽  
A Value ◽  
Available Information ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Covalent Complex

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

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