Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase

dc.contributor.author	Labrou, NE	en
dc.contributor.author	Eliopoulos, E	en
dc.contributor.author	Clonis, YD	en
dc.date.accessioned	2014-06-06T06:43:11Z
dc.date.available	2014-06-06T06:43:11Z
dc.date.issued	1996	en
dc.identifier.issn	02646021	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/1078
dc.relation.uri	http://www.scopus.com/inward/record.url?eid=2-s2.0-0030000366&partnerID=40&md5=2abc0108def79b2ef9e1ae705c12eb47	en
dc.subject.other	amide	en
dc.subject.other	anthraquinone	en
dc.subject.other	carboxyl group	en
dc.subject.other	cibacron blue f3ga	en
dc.subject.other	dye	en
dc.subject.other	malate dehydrogenase	en
dc.subject.other	oxaloacetic acid	en
dc.subject.other	oxoacid	en
dc.subject.other	reduced nicotinamide adenine dinucleotide	en
dc.subject.other	animal cell	en
dc.subject.other	article	en
dc.subject.other	binding affinity	en
dc.subject.other	cattle	en
dc.subject.other	chromatophore	en
dc.subject.other	competitive inhibition	en
dc.subject.other	controlled study	en
dc.subject.other	enzyme active site	en
dc.subject.other	enzyme binding	en
dc.subject.other	enzyme inactivation	en
dc.subject.other	enzyme inhibition	en
dc.subject.other	heart mitochondrion	en
dc.subject.other	hydrophobicity	en
dc.subject.other	molecular model	en
dc.subject.other	nonhuman	en
dc.subject.other	priority journal	en
dc.subject.other	spectrometry	en
dc.subject.other	titrimetry	en
dc.subject.other	Affinity Labels	en
dc.subject.other	Animals	en
dc.subject.other	Anthraquinones	en
dc.subject.other	Binding Sites	en
dc.subject.other	Cattle	en
dc.subject.other	Coloring Agents	en
dc.subject.other	Drug Design	en
dc.subject.other	Enzyme Inhibitors	en
dc.subject.other	Ligands	en
dc.subject.other	Malate Dehydrogenase	en
dc.subject.other	Mitochondria, Heart	en
dc.subject.other	Models, Molecular	en
dc.subject.other	Triazines	en
dc.subject.other	Animalia	en
dc.subject.other	Bos taurus	en
dc.subject.other	Bovinae	en
dc.title	Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase	en
heal.type	journalArticle	en
heal.publicationDate	1996	en
heal.abstract	Molecular modelling and kinetic inhibition studies, as well as K(D) determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower K(D) values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an α-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (K(D) 1.3 μM, which corresponded to a 219-fold decrease over the K(D) of a control dye). BM5 displayed competitive inhibition with respect to both NADH (K(i) 2.7 μM) and oxaloacetate (K(i) 9.6 μM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions.	en
heal.journalName	Biochemical Journal	en
dc.identifier.issue	2	en
dc.identifier.volume	315	en
dc.identifier.spage	695	en
dc.identifier.epage	703	en