ABSTRACT
Enantioselectivity of enzyme catalysis is often rationalized via active site models. These models are constructed on the basis of comparing the enantiomeric excess of product observed in a series of reactions which are conducted with a range of homologous substrates, typically carrying various side chain substitutions. Surprisingly the practical application of these simple but informative 'pocket size' models has been rarely tested in genetic engineering experiments. In this paper we report the construction, purification and enantioselectivity of two recombinant Rhizomucor miehei lipases which were designed to check the validity of such a model in reactions of ring opening of oxazolin-5(4H)-ones.
Subject(s)
Lipase/metabolism , Oxazoles/metabolism , Rhizomucor/enzymology , Catalytic Domain , Cloning, Molecular , Computer Simulation , Fungal Proteins , Hydrogen Bonding , Kinetics , Lipase/genetics , Models, Molecular , Mutagenesis, Site-Directed , Oxazoles/chemistry , Oxazolone , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , StereoisomerismABSTRACT
Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355-366.
Subject(s)
Antigens, Viral/chemistry , Glycoproteins/chemistry , Hepacivirus/chemistry , Membrane Proteins , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Antigens, CD/metabolism , Binding Sites , Computer Simulation , Dimerization , Heparin/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Receptors, Virus/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Surface Properties , Tetraspanin 28ABSTRACT
Lipase enzymes have found increasingly widespread use, especially in biotransformation reactions in organic synthesis. Due to their efficiency and high enantioselectivity, they can be employed in a variety of reactions to carry out asymmetric hydrolyses, esterifications and transesterifications. However, the reasons for their stereospecificity have not been fully correlated with the enzyme structure. Employing molecular modelling techniques and existing experimental data, a transesterification reaction using Rhizomucor miehei lipase was studied. The results indicate that the major controlling factor for this reaction is hydrophobic in nature, providing support for previous literature hypotheses. In addition, computational experiments suggest that the origin of enantioselectivity is the formation of essential hydrogen bonds in and around the catalytic triad of active site residues. Only one enantiomer of the substrate is able to form these hydrogen bonds during the formation of the first tetrahedral transition state.
