Enzyme–Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

Biochemistry ◽  
2015 ◽  
Vol 54 (40) ◽  
pp. 6176-6185 ◽  
Author(s):  
Johannes P. M. Schelvis ◽  
Xuling Zhu ◽  
Yvonne M. Gindt
Keyword(s):  
Substrate Binding ◽  
Binding Kinetics ◽  
Enzyme Substrate ◽  
Thymidine Dimer

scholarly journals Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power

2020 ◽  
Vol 16 (1) ◽  
pp. e1007600 ◽  
Author(s):  
Vaitea Opuu ◽  
Giuliano Nigro ◽  
Thomas Gaillard ◽  
Emmanuelle Schmitt ◽  
Yves Mechulam ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Adaptive Landscape ◽  
Enzyme Substrate ◽  
Catalytic Power

Steps in the reactions of proteolytic enzymes with their substrates

1970 ◽  
Vol 257 (813) ◽  
pp. 105-110 ◽  
Keyword(s):  
Active Sites ◽  
Dissociation Constants ◽  
Proteolytic Enzymes ◽  
Substrate Binding ◽  
Hydrolytic Enzymes ◽  
Dimensional Structure ◽  
Chemical Information ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Wide Range

Kinetic experiments should be designed to answer specific questions about a reaction mechanism. The present paper is intended to show how a number of specific questions have been answered. Chymotrypsin and trypsin are mainly used to illustrate the different approaches, but many of the arguments used are equally applicable to the reactions of other hydrolytic enzymes with serine-OH or cysteine-SH at the active site. T he recognition of serine-OH and cysteine-SH as essential groups at the active sites of different hydrolytic enzymes did not rest on kinetic evidence. This was deduced from the correlation of enzyme activity with the extent of modification of specially reactive groups. The investigation of proton dissociation equilibria and the assignment of dissociation constants to groups with specified functions in substrate binding, catalysis or protein conformation was the first objective of serious kinetic studies of enzyme reactions. Steady state rate measurements over a wide range of pH showed that groups with p K 6.25 and 6.85 respectively are involved in the catalytic activity of trypsin and chymotrypsin with certain specific substrates (Hammond & Gutfreund 1955). In the case of chymotrypsin it was also shown by Hammond & Gutfreund (1955) that a group with a more alkaline pK is involved in substrate binding. This latter group was subsequently identified and its function was elucidated through the elegant experiments of Oppenheimer, Labouresse & Hess (1966). The identification of histidine as the group with p K A near neutrality, involved in the catalytic mechanism of trypsin and chymotrypsin, was subsequently confirmed by direct chemical methods by Schoelmann & Shaw (1963). Only kinetic analysis can demonstrate the involvement of proton donors or acceptors with specific properties in enzyme-substrate interaction or in catalysis. The clear identification of chemical groups capable of performing such functions is coming from the crystallographic analysis of the three-dimensional structure at the site of enzyme-substrate interaction, as illustrated in other papers presented in this discussion. Very interesting chemical information is obtained when the effect of structure on reactivity is synthesized from the composite of crystallographic and kinetic data.

3β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN THE HUMAN FOETAL ADRENAL CORTEX

1965 ◽  
Vol 48 (3) ◽  
pp. 423-428 ◽  
Author(s):  
M. Niemi ◽  
A. H. Baillie
Keyword(s):  
Dehydrogenase Activity ◽  
Adrenal Cortex ◽  
Substrate Binding ◽  
Hydroxysteroid Dehydrogenase ◽  
List Type ◽  
Histochemical Reaction ◽  
Enzyme Substrate ◽  
Human Male ◽  
Foetal Life ◽  
Free Steroid

ABSTRACT 3β-Hydroxysteroid dehydrogenase activity was studied histochemically in the adrenal cortex of ten human male foetuses, ranging in crownrump length from 3.0 cm to 18.3 cm, with the following steroids: 3β-hydroxy-pregn-5-en-20-one (pregnenolone). 3β,17α-dihydroxy-pregn-5-en-20-one (17α-hydroxypregnenolone). 3β-hydroxy-androst-5-en-17-one (DHA). 3β,17β-dihydroxy-androst-5-ene (androstenediol). 3β-sulphoxy-pregn-5-en-20-one (pregnenolone sulphate). 3β-sulphoxy-17α-hydroxy-pregn-5-en-20-one (17α-hydroxy-pregnenolone sulphate) 3β-sulphoxy-androst-5-en-17-one (DHAsulphate). 3β-hydroxy-5α-androstan-17-one (epiandrosterone). After incubation with pregnenolone, 17α-hydroxypregnenolone, DHA and androstenediol a positive histochemical reaction was obtained in the inner part of the »definitive« cortex and throughout the foetal cortex of all adrenals studied. Initially very weak, the reaction became strongly positive about the twelfth week of foetal life. Pregnenolone sulphate and 17α-hydroxypregnenolong sulphate also gave a histochemical reaction in all the adrenals investigated, but DHA sulphate differed significantly from the free steroid by giving a very poor reaction. Formazan deposition followed incubation with epiandrosterone in all adrenals used and this may imply that a δ5 configuration is not necessary for enzyme-substrate binding.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

Engineering of a keto acid reductase through reconstructing the substrate binding pocket to improve its activity

2019 ◽  
Vol 9 (8) ◽  
pp. 1961-1969 ◽  
Author(s):  
Di-Chen Wang ◽  
Heng Li ◽  
Shu-Ning Xia ◽  
Ya-Ping Xue ◽  
Yu-Guo Zheng
Keyword(s):  
Point Mutation ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Keto Acid ◽  
Substrate Binding Pocket ◽  
Substrate Docking ◽  
Enzyme Substrate ◽  
Phenylbutyric Acid

Enzyme–substrate docking-guided point mutation of the substrate-binding pocket to generate mutant L244G/A250G/L245R with superior activity in the synthesis of (R)-2-hydroxy-4-phenylbutyric acid.

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