scholarly journals A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1970 ◽  
Vol 117 (1) ◽  
pp. 169-176 ◽  
Author(s):  
W. G. Bardsley ◽  
C. M. Hill ◽  
R. W. Lobley
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Enzyme Preparation ◽  
Diamine Oxidase ◽  
Substrate Binding ◽  
Pig Kidney ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Rf Values

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with RF values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme–substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0–9.0 Å from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.

scholarly journals Synthesis and oxidation of aminoalkyl-onium compounds by pig kidney diamine oxidase

1971 ◽  
Vol 122 (4) ◽  
pp. 557-567 ◽  
Author(s):  
W. G. Bardsley ◽  
J. S. Ashford ◽  
C. M. Hill
Keyword(s):  
Substrate Concentration ◽  
Diamine Oxidase ◽  
Amino Group ◽  
High Substrate Concentration ◽  
High Substrate ◽  
Pig Kidney ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Potent Inhibition

1. The preparation of a series of compounds derived from diamines by replacing one amino group by a dimethylsulphonium, isothiuronium, trimethylammonium, NN′-dimethylimidazolium or N-methylpyridinium species is described. 2. The behaviour of these compounds as substrates of pig kidney diamine oxidase is reported. All but the trimethylammonium compounds proved to be substrates. 3. Many of these compounds showed potent inhibition at high substrate concentration and this was studied. 4. On the basis of these and other observations a scheme for enzyme–substrate interaction is suggested.

scholarly journals Characteristics and composition of the vitamin K-dependent γ-glutamyl carboxylase-binding domain on osteocalcin

2002 ◽  
Vol 364 (1) ◽  
pp. 323-328 ◽  
Author(s):  
Roger J.T.J. HOUBEN ◽  
Dirk T.S. RIJKERS ◽  
Thomas B. STANLEY ◽  
Francine ACHER ◽  
Robert AZERAD ◽  
...  
Keyword(s):  
Binding Site ◽  
Vitamin K ◽  
Active Site ◽  
Substrate Binding ◽  
Direct Interaction ◽  
Binding Domain ◽  
High Affinity ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Gla Domain

Two different sites on vitamin K-dependent γ-glutamyl carboxylase (VKC) are involved in enzyme—substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-Km substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the γ-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.

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.

scholarly journals Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

2011 ◽  
Vol 77 (16) ◽  
pp. 5730-5738 ◽  
Author(s):  
Hanna M. Dudek ◽  
Gonzalo de Gonzalo ◽  
Daniel E. Torres Pazmiño ◽  
Piotr Stępniak ◽  
Lucjan S. Wyrwicz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Active Site ◽  
Structural Model ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

scholarly journals Thermodynamics of the DNA Repair Process by Endonuclease VIII

Acta Naturae ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 29-37 ◽  
Author(s):  
O. A. Kladova ◽  
N. A. Kuznetsov ◽  
O. S. Fedorova
Keyword(s):  
Activation Enthalpy ◽  
Substrate Binding ◽  
Repair Process ◽  
Molecular Processes ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Different Temperatures ◽  
Enthalpy And Entropy ◽  
Transition Complex ◽  
Dna Substrate

In the present work, a thermodynamic analysis of the interaction between endonuclease VIII (Endo VIII) and model DNA substrates containing damaged nucleotides, such as 5,6-dihydrouridine and 2-hydroxymethyl-3-hydroxytetrahydrofuran (F-site), was performed. The changes in the fluorescence intensity of the 1,3-diaza-2-oxophenoxazine (tCO) residue located in the complementary chain opposite to the specific site were recorded in the course of the enzyme-substrate interaction. The kinetics was analyzed by the stopped-flow method at different temperatures. The changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage, were calculated. The comparison of the kinetic and thermodynamic data characterizing the conformational transitions of enzyme and DNA in the course of their interaction made it possible to specify the nature of the molecular processes occurring at the stages of substrate binding, recognition of the damaged base, and its removal from DNA.

scholarly journals Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 α-demethylase from maize (Zea mays) seedlings

1991 ◽  
Vol 277 (2) ◽  
pp. 483-492 ◽  
Author(s):  
M Taton ◽  
A Rahier
Keyword(s):  
Zea Mays ◽  
Substrate Specificity ◽  
Binding Site ◽  
Critical Role ◽  
Biochemical Properties ◽  
Product Specificity ◽  
Enzyme Substrate ◽  
Absolute Requirement ◽  
High Degree ◽  
Cytochrome P 450

The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.

scholarly journals Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

2012 ◽  
Vol 287 (11) ◽  
pp. 7925-7934 ◽  
Author(s):  
Magnus Monné ◽  
Daniela Valeria Miniero ◽  
Lucia Daddabbo ◽  
Alan J. Robinson ◽  
Edmund R. S. Kunji ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Substrate Binding ◽  
Single Mutation ◽  
Substrate Binding Site
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