MOLECULAR MODELING FOR THE DESIGN OF CHIMERIC BIOMIMETIC DYE-LIGANDS AND THEIR INTERACTION WITH BOVINE HEART MITOCHONDRIAL MALATE-DEHYDROGENASE

Molecular modelling and kinetic inhibition studies, as well as K-D det erminations by both difference-spectra and enzyme-inactivation studies , were employed to assess the ability of purpose-designed chimaeric bi omimetic dyes (BM dyes) to act as affinity ligands for bovine heart L- malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-rec ognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the s ubstrate of MDH. The chromophore anthraquinone moiety remained unchang ed and the same as that of the parent dye Vilmafix Blue A-R (VBAR), re cognizing the nucleotide-binding site of MDH. The monochlorotriazine B M dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accom panied by a characteristic spectral change in the range 500-850 nm. Th is phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-b iomimetic anthraquinone dyes, all BM dyes exhibited lower K-D values a nd therefore higher affinity for MDH. The enzyme bound preferably to B M ligands substituted with a biomimetic aromatic moiety bearing an alp ha-keto acid group and an amide linkage, rather than a monocarboxyl gr oup. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (K-D 1.3 mu M, which corresponded to a 219-fold decrease over the K-D of a control dye). BM5 displayed competitive inhibition with respect to bot h NADH (K-i 2.7 mu M) and oxaloacetate (K-i 9.6 mu M). A combination o f molecular modelling and experimental studies has led to certain conc lusions. The positioning of the dye in the enzyme is primarily achieve d by the recognition and positioning of the nucleotide-pseudomimetic a nthraquinone moiety. The hydrophobic groups of the dye provide the dri ving force for positioning of the ketocarboxyl biomimetic moiety. A ma tch between the alternating polar and hydrophobic regions of the enzym e binding site with those of the biomimetic moiety is desirable. The l ength of the biomimetic moiety should be conserved in order for the ke to acid to approach the enzyme active site and form charge-charge inte ractions.