pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger

2000 ◽  
Vol 349 (2) ◽  
pp. 623-628 ◽  
Author(s):  
Ulla CHRISTENSEN
Keyword(s):  
Aspergillus Niger ◽  
Ph Dependence ◽  
Acid Group ◽  
Reaction Scheme ◽  
Free Enzyme ◽  
Pka Values ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
State Kinetic

The presteady-state kinetic parameters of the interaction of wild-type glucoamylase from Aspergillus niger (EC 3.2.1.3) with maltose were obtained and analysed in the pH range 3-7 with intervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. E+S ES1 ES2 E+P The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pKa values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pKa 6.5 had no effect on k2 (1640 s-1) or k-2 (20±4 s-1), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K1 from 40 to 6.3 mM. In other words, when the substrate is bound, the pKa of the acid group changes to increase the fraction of reactive enzyme. Since this pKa parallels that of the Michaelis complex, known from the pH-dependence of kcat, the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pKa = 4.75 group leads to a small decrease in k2 to 1090 s-1, and also to minor changes in K1. The group with pKa = 2.7 leads to a major decrease of k2, of which the limit may be zero, but shows no effect on K1. Thus no difference is seen between the pKa values of the free enzyme and of the first enzyme-substrate complex at low pH.

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

1967 ◽  
Vol 45 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Harvey Kaplan ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Free Enzyme ◽  
State Equations ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Special Cases ◽  
Influence Of Ph

General steady-state equations are worked out for enzyme reactions which occur according to the scheme [Formula: see text]Equations showing the pH dependence of the kinetic parameters are developed in a form which distinguishes between essential and nonessential ionizing groups. The pK dependence of [Formula: see text], the second-order constant extrapolated to zero substrate constant, gives pK values for groups which ionize on the free enzyme, but reveals such a pK only if the corresponding group is also involved in the breakdown of the Michaelis complex. General steady-state equations are also developed for the case in which an inhibitor can combine with the free enzyme, the enzyme–substrate complex, and also a second intermediate (e.g. an acyl enzyme). The equations are given in a form that is convenient for analyzing the experimental results, and a number of special cases are considered. It is shown how the type of inhibition depends not only on the nature of the inhibitor but also on that of the substrate, an important factor being the rate-determining step of the reaction. Examples of the various kinds of behavior are given.

scholarly journals The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli

1968 ◽  
Vol 106 (2) ◽  
pp. 455-460 ◽  
Author(s):  
D. R. Trentham ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ph Dependence ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Nitrophenyl Phosphate ◽  
Steady State Rate ◽  
State Rate ◽  
Rate Of Hydrolysis ◽  
Ph Range

1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5·5–8·5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8·1 and 5·9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8·1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5·9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme–substrate complex.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

2019 ◽  
pp. 1-7
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Complex Formation ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Continuous Formation ◽  
Enzyme Substrate ◽  
The Difference ◽  
Total Enzyme

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration. Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters. Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

Final Phase of Enzyme Reactions Following a Michaelis-Menten Mechanism in which the Free Enzyme and/or the Enzyme-Substrate Complex are Unstable

1994 ◽  
Vol 375 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Ramón Varón ◽  
Carmelo Garrido del Solo ◽  
Manuela Garcίa-Moreno ◽  
Angela Sánchez-Gracia ◽  
Francisco Garcίa-Cánovas
Keyword(s):  
Free Enzyme ◽  
Final Phase ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Enzyme Substrate

BTX Degradation: The concept of microbial integration

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Gas Chromatography ◽  
Soil Sample ◽  
High Activity ◽  
Niger Delta ◽  
Batch Reactor ◽  
Free Enzyme ◽  
Delta Area ◽  
Substrate Complex ◽  
Fungal Activity ◽  
Enzyme Substrate

The concept of microbial integration was carried out to examine bacterial and fungal activity on bezene, toluence and xylene (BTX) degradation in a batch reactor. The investigation was conducted for thirty five day of exposure of contact of members and substrate which yielded enzyme substrate complex as well disintegrated to produce products and free enzyme. Bacterial and fungal concentration was monitored per week and the results obtained recorded. The gas chromatography results of Ngara soil sample investigated reveals the concentration of M, P, and O – Xylene for different days of exposure. Increase in both bacterial and fungal was experienced with decrease in BTX concentration, whereas increase in bacterial is more than fungi, indicating the high activity of bacterial in the reactor than that of fungi. Although, both were well integrated in bioremediation program to enhance the effective remediation of BTX contaminants in Ngara soil, Omuigwe Alun Community, Niger Delta Area of Nigeria.

scholarly journals Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable

1993 ◽  
Vol 294 (2) ◽  
pp. 459-464 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
R Varón-Castellanos
Keyword(s):  
Data Analysis ◽  
Experimental Design ◽  
Numerical Simulations ◽  
Kinetic Analysis ◽  
Kinetic Data ◽  
Time Course ◽  
Enzyme Concentration ◽  
Free Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate

A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended.

scholarly journals Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

2003 ◽  
Vol 370 (1) ◽  
pp. 331-337 ◽  
Author(s):  
Paloma SÁNCHEZ-TORRES ◽  
Jaap VISSER ◽  
Jacques A.E. BENEN
Keyword(s):  
Aspergillus Niger ◽  
Conformational Change ◽  
Three Dimensional ◽  
Pectin Lyase ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Fluorescence Studies ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Positively Charged

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed β-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689] and the modelled enzyme—substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762—8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154→Arg and Asp154→Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme—substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the pKa of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186—Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689]. Substitution of Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186→Asn/Asp221→Asn enzyme appeared less stable than the wild-type enzyme, even at pH6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp186—Asp221 pair.

scholarly journals The pH-dependence of pepsin-catalysed reactions

1969 ◽  
Vol 113 (2) ◽  
pp. 353-362 ◽  
Author(s):  
A. J. Cornish-Bowden ◽  
J. R. Knowles
Keyword(s):  
Continuous Monitoring ◽  
Ph Dependence ◽  
Preceding Paper ◽  
Free Enzyme ◽  
Peptide Substrates ◽  
Pka Values ◽  
Hydrolysis Of

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pKa values of 1·1 and 3·5 in the plots of k0/Km versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pKa values of 1·0 and 4·7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pKa values of 1·0 and 4·7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pKa values of 3·5 for the dependence of k0/Km for these materials.

scholarly journals The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

2019 ◽  
pp. 1-12
Author(s):  
Ikechukwu I. Udema ◽  
Abraham Olalere Onigbinde
Keyword(s):  
Maximum Velocity ◽  
Order Rate Constant ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Coefficient Of Determination ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
The Relationship

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) which accounts for sequestration. Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [ST] <  Michaelis-Menten constant, KM. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [EST] and the enzyme-substrate complex ([ES]) prepared for product, P, formation. Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [ST] (if v/[ST] is constant with coefficient of determination = 1), then KM is not applicable.

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