scholarly journals Mechanistic studies of carboxypeptidase Y from Saccharomyces cerevisiae. pH and pD profiles and inactivation at low pH (pD) values

1980 ◽  
Vol 187 (3) ◽  
pp. 843-849 ◽  
Author(s):  
W T Chang ◽  
K T Douglas
Keyword(s):  
Tertiary Structure ◽  
Low Ph ◽  
Carboxypeptidase Y ◽  
Mechanistic Studies ◽  
Ph Profile ◽  
First Order ◽  
Steady State Kinetics ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Hydrolysis Of

Steady-state kinetics of carboxypeptidase Y, a proteinase from yeast, were studied by using the reaction of 4-nitrophenyl trimethylacetate as a probe. The pH profile of kcat. is sigmoidal in H2O-based buffers for the carboxypeptidase Y-catalysed hydrolysis of this ester (kcat. referring to the rate of deacylation of trimethylacetyl-carboxypeptidase Y). The corresponding pD profile in 2H2O is doubly sigmoidal, with inflexions at pD approximately 3.8 and approximately 6.8. The ionization of pKDapp. approximately 3.8 is caused by a rapid inactivation in 2H2O media by a process that is only slowly reversed on transfer to pH 7.00 phosphate buffer in H2O. The corresponding inactivation in H2O-based buffers of low pH is considerably slower (approximately 30-fold), follows a first-order rate-dependence and is very strongly pH-dependent, indicating some form of co-operative change in enzyme tertiary structure.

Iodoacetate inactivation of rape alcohol dehydrogenase

1980 ◽  
Vol 45 (5) ◽  
pp. 1601-1607 ◽  
Author(s):  
Marie Stiborová ◽  
Sylva Leblová
Keyword(s):  
Alcohol Dehydrogenase ◽  
Protein Molecule ◽  
Sulfhydryl Groups ◽  
Inactivation Rate ◽  
First Order ◽  
Ph Dependent ◽  
Kinetics Of

Iodoacetate inactivates rape alcohol dehydrogenase (ADH, EC 1.1.1.1). The inactivation rate follows the kinetics of the first order, is pH-dependent, and decreases below pH 7.5. Besides irreversible alkylation of the sulfhydryl groups of the enzyme iodoacetate also forms a reversible complex with rape ADH. The coenzyme (NAD) and its analogs (ATP, ADP, AMP) competitively protect the enzyme against alkylation; o-phenanthroline also protects the enzyme against alkylation yet noncompetitively with respect to iodoacetate. Imidazole and o-phenanthroline compete with one another for binding to the protein molecule of rape ADH. Whereas o-phenanthroline decreases the inactivation rate imidazole increases the rate of iodoacetate inactivation.

Steady-state and pre-steady-state kinetics of the mitochondrial F(1)F(o) ATPase: is ATP synthase a reversible molecular machine?

2000 ◽  
Vol 203 (1) ◽  
pp. 41-49 ◽  
Author(s):  
A.D. Vinogradov
Keyword(s):  
Steady State ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Gamma Subunit ◽  
Change Mechanism ◽  
Steady State Kinetics ◽  
Energy Dependent ◽  
Variable Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of

H(+)-ATP synthase (F(1)F(o) ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F(1), which is ideally suited for the alternating binding change mechanism, with a rotating gamma-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (P(i)) and Mg(2+) participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F(1) and F(1)F(o) demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, P(i), Mg(2+) and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F(1)F(o) ATPase is proposed.

Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride

1977 ◽  
Vol 55 (1) ◽  
pp. 19-26 ◽  
Author(s):  
R. James Maguire
Keyword(s):  
Steady State ◽  
Trichoderma Viride ◽  
Solution Temperature ◽  
Temperature Range ◽  
A Value ◽  
Steady State Kinetics ◽  
Decreasing Ph ◽  
Ph Curve ◽  
Kinetics Of ◽  
Hydrolysis Of

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol–buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 × 10−3 M to 9 × 10−2 M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate – pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol−1 over the temperature range 5–56 °C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol.The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-β-D-glucoside was examined by pre-steady-state methods in which [Formula: see text], and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 s−1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism [Formula: see text]) (ii) a log rate–pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5–6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol−1 at pH 5.7 over the temperature range 10–60 °C.

Evidence for the formation of lignin-hexenuronic acid-xylan complexes during modified kraft pulping processes

Holzforschung ◽  
2006 ◽  
Vol 60 (2) ◽  
pp. 137-142 ◽  
Author(s):  
Zhi-Hua Jiang ◽  
Jean Bouchard ◽  
Richard Berry
Keyword(s):  
Acid Hydrolysis ◽  
Kraft Pulp ◽  
Kraft Pulping ◽  
Order Kinetic ◽  
Kraft Pulps ◽  
First Order ◽  
Pseudo First Order ◽  
Hexenuronic Acid ◽  
Kinetics Of ◽  
Hydrolysis Of

Abstract The finding that hexenuronic acid (HexA) groups can be selectively removed from kraft pulps by acid hydrolysis has provided an opportunity to reduce bleaching chemicals. However, there is evidence that the acid hydrolysis is not uniform. In this report, we evaluate the kinetics of acid hydrolysis of HexA in a xylan sample enriched with HexA, a conventional kraft pulp, and three modified kraft pulps: anthraquinone pulp (Kraft-AQ), polysulfide pulp (PS), and polysulfide-anthraquinone pulp (PS-AQ). We found that HexA present in the xylan and conventional kraft pulp behaved similarly toward the acid hydrolysis throughout. On the other hand, HexA present in the Kraft-AQ, PS-AQ and PS pulps was heterogeneous toward acid hydrolysis and the reaction can be separated into two pseudo-first-order kinetic phases, each of which has a different rate constant. The kinetic data provide evidence for the formation of lignin-HexA-xylan complexes during modified kraft pulping processes.

Steady-State Kinetics of Enzyme Reactions in the Presence of Added Nucleophiles

1972 ◽  
Vol 50 (12) ◽  
pp. 1334-1359 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Specific Binding ◽  
Specific Binding Sites ◽  
Steady State Kinetics ◽  
Special Cases ◽  
Kinetics Of Formation ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Concentration Graphs ◽  
Kinetics Of ◽  
Hydrolysis Of

Many enzyme-catalyzed reactions, such as hydrolyses, give rise to two products P1 and P2 which are formed in different reaction steps. The second product P2 is frequently formed by hydrolysis of an intermediate such as an acyl-enzyme or a phosphoryl-enzyme. An alternative nucleophile N introduced into the system forms an additional product P3. The present paper is concerned with the kinetics of formation of P1, P2, and P3 in the presence of added nucleophiles. A number of alternative mechanisms are considered, and equations are derived for the rates of formation of the three products, and the Michaelis constant, as functions of nucleophile concentration. Graphs are presented showing the variations of these parameters with the concentration of N, for a variety of special cases. Special attention is given to the possibility of specific binding sites for the water and the nucleophile molecules.The data for a number of enzyme systems are discussed with reference to the treatment. For reactions catalyzed by alkaline phosphatase it is concluded that only one mechanism (mechanism VI) is consistent with the results.

scholarly journals The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

1983 ◽  
Vol 211 (1) ◽  
pp. 237-242 ◽  
Author(s):  
G B Irvine ◽  
N L Blumsom ◽  
D T Elmore
Keyword(s):  
Cathepsin D ◽  
Order Rate Constant ◽  
Chicken Liver ◽  
Lag Phase ◽  
Hydrophilic Group ◽  
Steady State Kinetics ◽  
Cationic Group ◽  
Specificity Constant ◽  
Kinetics Of ◽  
Hydrolysis Of

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.

KINETIC STUDIES ON THE HYDROLYSIS OF BENZOYLCHOLINE BY HUMAN SERUM CHOLINESTERASE

1956 ◽  
Vol 34 (1) ◽  
pp. 637-653 ◽  
Author(s):  
W. Kalow ◽  
K. Genest ◽  
N. Staron
Keyword(s):  
Kinetic Studies ◽  
Reaction Rates ◽  
Serum Cholinesterase ◽  
Initial Reaction ◽  
Ultraviolet Spectrophotometry ◽  
First Order ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

Benzoylcholine stands out from other known substrates of serum cholinesterase because of its high apparent affinity for this enzyme combined with a rapid rate of destruction. The reaction kinetics of the hydrolysis of benzoylcholine can be studied by ultraviolet spectrophotometry, since the absorbance decreases in proportion to the concentration of substrate. Kinetic data obtained by measuring initial reaction rates, and by analyzing continuous hydrolysis curves, are the same within the range of experimental error. The enzymatic data are compatible with the assumption that in the presence of high substrate concentrations a complex consisting of esterase and two substrate molecules is formed. This complex is hydrolyzed more slowly than the complex containing one molecule of substrate which is formed at low concentrations of benzoylcholine. Alkaline hydrolysis of benzoylcholine follows the kinetics of a first order reaction.

Sign in / Sign up

Export Citation Format

Share Document