The Kinetic Basis of a General Method for the Investigation of Active Site Content of Enzymes and Catalytic Antibodies: First-Order Behaviour under Single-turnover and Cycling Conditions

2000 ◽  
Vol 204 (2) ◽  
pp. 239-256 ◽  
Author(s):  
CHRISTOPHER M. TOPHAM ◽  
SHERAZ GUL ◽  
MARINA RESMINI ◽  
SANJIV SONKARIA ◽  
GERRARD GALLACHER ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Antibodies ◽  
First Order ◽  
Single Turnover ◽  
General Method

Towards the Temporal Approach to Abstract Data Types

1988 ◽  
Vol 11 (1) ◽  
pp. 49-63
Author(s):  
Andrzej Szalas
Keyword(s):  
Temporal Logic ◽  
Abstract Data Types ◽  
Data Types ◽  
First Order ◽  
Abstract Data ◽  
Order Completeness ◽  
General Method

In this paper we deal with a well known problem of specifying abstract data types. Up to now there were many approaches to this problem. We follow the axiomatic style of specifying abstract data types (cf. e.g. [1, 2, 6, 8, 9, 10]). We apply, however, the first-order temporal logic. We introduce a notion of first-order completeness of axiomatic specifications and show a general method for obtaining first-order complete axiomatizations. Some examples illustrate the method.

scholarly journals Mechanistic and active-site studies on d(–)-mandelate dehydrogenase from Rhodotorula graminis

1992 ◽  
Vol 281 (1) ◽  
pp. 211-218 ◽  
Author(s):  
D P Baker ◽  
C Kleanthous ◽  
J N Keen ◽  
E Weinhold ◽  
C A Fewson
Keyword(s):  
Active Site ◽  
Complete Sequence ◽  
Histidine Residue ◽  
Extended Version ◽  
Order Process ◽  
First Order ◽  
Tryptic Digest ◽  
Complete Inactivation ◽  
Hydrolysis Of ◽  
High Voltage Electrophoresis

D(–)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(–)-mandelate to phenylglyoxylate. D(–)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [‘D(–)-bromoacetylmandelic acid’], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(–)-Mandelate dehydrogenase was inactivated by D(–)-bromoacetylmandelate in a psuedo-first-order process. D(–)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(–)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(–)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(–)-bromoacetylmandelate reacts. D(–)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(–)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

scholarly journals Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

2013 ◽  
Vol 288 (22) ◽  
pp. 15532-15536 ◽  
Author(s):  
Natalie Gumataotao ◽  
Misty L. Kuhn ◽  
Natalia Hajnas ◽  
Richard C. Holz
Keyword(s):  
Active Site ◽  
Nitrile Hydratase ◽  
Stopped Flow ◽  
Single Turnover

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

scholarly journals Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding

1996 ◽  
Vol 314 (3) ◽  
pp. 985-991 ◽  
Author(s):  
Subrata ADAK ◽  
Abhijit MAZUMDER ◽  
Ranajit K. BANERJEE
Keyword(s):  
Active Site ◽  
Native Enzyme ◽  
Tyrosine Residue ◽  
Tyrosine Residues ◽  
First Order ◽  
First Order Kinetics ◽  
Aromatic Donor ◽  
Pseudo First Order ◽  
Modified Enzyme ◽  
Compound Ii

The plausible role of arginine and tyrosine residues at the active site of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate constants of 24 M-1·min-1, 0.8 M-1·min-1 and 0.54 M-1·min-1 respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0 M-1·min-1. The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement of arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (∆G´) for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete inactivation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35 mM compared with 10 mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichroism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

A Circular Inclusion With Circumferentially Inhomogeneous Sliding Interface in Plane Elastostatics

1998 ◽  
Vol 65 (1) ◽  
pp. 30-38 ◽  
Author(s):  
C. Q. Ru
Keyword(s):  
Circular Inclusion ◽  
Form Solution ◽  
Infinite Matrix ◽  
Rigorous Solution ◽  
First Order ◽  
Sliding Interface ◽  
The Mean ◽  
Plane Elastostatics ◽  
Basic Boundary ◽  
General Method

A general method is presented to obtain the rigorous solution for a circular inclusion embedded within an infinite matrix with a circumferentially inhomogeneous sliding interface in plane elastostatics. By virtue of analytic continuation, the basic boundary value problem for four analytic functions is reduced to a first-order differential equation for a single analytic function inside the circular inclusion. The finite form solution is obtained that includes a finite number of unknown constants determined by the analyticity of the solution and certain other auxiliary conditions. With this method, the exact values of the average stresses within the circular inclusion can be calculated without solving the full problem. Several specific examples are used to illustrate the method. The effects of the circumferential variation of the interface parameter on the mean stress at the interface and the average stresses within the inclusion are discussed.

A NEW FAMILY OF FIRST-ORDER TIME-DELAYED CHAOTIC SYSTEMS

2011 ◽  
Vol 21 (09) ◽  
pp. 2547-2558 ◽  
Author(s):  
XIAOMING ZHANG ◽  
JUFANG CHEN ◽  
JIANHUA PENG
Keyword(s):  
Chaotic Systems ◽  
Linear Time ◽  
Period Doubling ◽  
First Order ◽  
New Family ◽  
Delayed Differential Equations ◽  
Stable Periodic ◽  
Delayed System ◽  
Bifurcation Direction ◽  
General Method

A general method for formulating first-order time-delayed chaotic systems with simple linear time-delayed term is proposed. The formulated systems are realized with electronic circuit experiments. In order to determine the unknown coefficients in a general delayed differential equations for having chaotic solutions, we follow the route of period-doubling bifurcation to chaos. Firstly, the conditions for a time-delayed system having a stable periodic solution, generating from a destablized steady state, is analyzed with Hopf bifurcation theory. Then the delay time parameter is changed according to the bifurcation direction to search the chaotic state, which is identified by the Lyapunov exponents spectra. The theoretical analysis is well confirmed by numerical simulations and circuit experiments.

scholarly journals A GENERAL METHOD FOR THE LABELING OF THE ACTIVE SITE OF ANTIBODIES AND ENZYMES

1959 ◽  
Vol 45 (10) ◽  
pp. 1470-1475 ◽  
Author(s):  
M. E. Koshland ◽  
F. Englberger ◽  
D. E. Koshland
Keyword(s):  
Active Site ◽  
General Method

scholarly journals Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

2021 ◽  
Author(s):  
Jaya Joshi ◽  
Qiang Li ◽  
Jorge D. Garcia-Garcia ◽  
Bryan J. Leong ◽  
You Hu ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Structure And Function ◽  
Comparative Genomic ◽  
Aerobic Conditions ◽  
Single Turnover ◽  
Metal Cofactor ◽  
Catalytic Metal ◽  
And Function ◽  
Sulfur Donor

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single–turnover suicide reaction that uses an active–site Cys residue as sulfur donor. Multiple turnover (i.e. catalytic) THI4s lacking an active–site Cys (non–Cys THI4s) that use sulfide as sulfur donor have been characterized—but only from archaeal methanogens that are anaerobic, O2–sensitivehyperthermophiles from sulfide–rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non–Cys THI4s in aerobic mesophiles from sulfide–poor habitats, suggesting that multiple–turnover THI4 operation is possible in aerobic, mild, low–sulfide conditions. This was confirmed by testing 23 representative non–Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were active, and more so when intracellular sulfidelevel was raised by supplying Cys, demonstrating that they function in the presence of O2 at mild temperatures and indicating they use sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non–Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2–sensitive methanogens but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

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