Multimeric enzymes

Kim, S.Y.et al. (2005) Novel type of enzyme multimerization enhances substrate affinity of oat P-glucosidase. J. Struct. Biol. 150, 1 10... 

A linear form of the Hill equation is used to evaluate the cooperative substrate-binding kinetics exhibited by some multimeric enzymes. The slope n, the Hill coefficient, reflects the number, nature, and strength of the interactions of the substrate-binding sites. A... 

The interpretation of much of the binding data given so far is based upon the assumption that the high affinity binding sites represent a population of independent sites. In the unphosphorylated II" " these sites would open up either to the periplas-mic or cytoplasmic side of the membrane independently of each other. The assumption ignores the evidence that the enzyme is, in fact, multimeric and that the data... 

Because mechanism-based inactivation depends on enzyme catalysis, there cannot be more than one molecule of inactivator bound to the enzyme active site. Thus formation of the covalent E-A species cannot result in a stoichiometry of inactivator to enzyme of greater than 1 1. In the case of multimeric enzymes, however, it may not be necessary to covalently modify all of the enzyme active sites within the multi-mer in order to effect total inactivation of the enzyme. In this situation one may observe a stoichiometry of less that 1 1. Under no circumstances, however, can a mechanism-based inactivator display a stoichiometry of greater than 1 1 with the enzyme. 

A novel nitrilase was purified from Aspergillus niger K10 cultivated on 2-cyanopyridine. It was found to be homologous to a putative nitrilase from Aspergillus fumigatus Af293. The nitrilase exhibited maximum activity at 45 °C and pH 8.0 with much less activity observed at slightly acid pH. Its substrate preference was for 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thio-phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. ( )-2-Phenylpropionitrile was only poorly converted by this enzyme and with minimal enantioselectivity. The enzyme was shown to be multimeric (>650 kDa) and be stabilized in the presence of sorbitol and xylitol [57]. 

The general types of protein-protein interactions that occur in cells include receptor-ligand, enzyme-substrate, multimeric complex formations, structural scaffolds, and chaperones. However, proteins interact with more targets than just other proteins. Protein interactions can include protein-protein or protein-peptide, protein-DNA/RNA or protein-nucleic acid, protein-glycan or protein-carbohydrate, protein-lipid or protein-membrane, and protein-small molecule or protein-ligand. It is likely that every molecule within a cell has some kind of specific interaction with a protein. 

Carpenter et al. [1.120] found that certain polymers (e. g. PVP) could stabilize multimeric enzymes during freezing and freeze drying by a different mechanism They cannot replace water molecules in the dried state therefore it is assumed that they inhibited the dissociation of the enzymes molecules induced by freezing and freeze drying. 

In addition to the binding of substrate (or in some cases co-substrates) at the active site, many enzymes have the capacity to bind regulatory molecules at sites which are usually spatially far removed from the catalytic site. In fact, allosteric enzymes are invariably multimeric (i.e. have a quaternary structure) and the allosteric (regulatory) sites are on different subunits of the protein to the active site. In all cases, the binding of the regulatory molecules is non covalent and is described in kinetic terms as noncompetitive inhibition. 

Pressure may cause several changes in enzymes, as well as some changes which are not directly associated with the catalytic process. These changes may include conformational changes and subunit dissociation-association processes. Pressures above 4000 bar may induce conformational changes to such an extent that the enzyme in effect becomes irreversibly denatured. These are dealt with in the next section. In this section we will deal with lower pressures and reversible processes, namely, interactions between subunits in quaternary structures. For most multimeric enzymes, the maintenance of... 

In a pressure study involving a multimeric enzyme, it will in general not be possible to decide how much of the effect is due to direct influence of pressure on the catalytic process and how much of it is due to indirect influence through subunit dissociation and accompanying deactivation. Generally, a self-association reaction may be expressed in either of two equivalent forms ... 

From a study involving several multimeric and monomeric enzymes, Penniston (1971) concluded that subunit dissociation must be considered as the major determinant of the effect of pressure on enzymic systems. This view may not be generally accepted. 

Enzymes may exist as simple monomers, or as homo- or heterodimers, or as multimers. In multimeric enzymes, each component monomer may possess a catalytic site alternatively, the catalytic site may be located at the interface between two or more monomers, or only one monomer of a heteromultimer may possess an active site. It is not uncommon in dimeric or multimeric enzymes containing two or more active sites for some degree of cooperativity to exist between the sites, with respect to the substrate binding or substrate turnover number (Monod et ah, 1965). 

This enzyme [EC 3.1.26.3], also known as RNase O and RNase D, catalyzes the endonucleolytic cleavage of RNA to 5 -phosphomonoesters. The enzyme cleaves multimeric tRNA precursors at the spacer region and is also involved in the processing of precursor rRNA, hnRNA, and early T7-mRNA. This enzyme can also act on double-stranded DNA. 

Viral structural proteins expressed by baculovirus infected insect cells assemble into multimeric structures that resemble viral core-like particles and virus-like particles (CLPs and VLPs, respectively). This presentation has brought the attention of researchers for the potential use of these structures as safe immunological reagents for virus or antibody detection in enzyme immuno assays, as vaccines, and more recently, as gene delivering systems for gene therapy [9]. 

It is not easy to mimic the shuffling of domains in vitro by manipulation of genes. For example, each catalytic polypeptide chain of the multimeric E. coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helixes. The E. coli enzyme ornithine transcarbamoylase (OTCase) is 32% identical in sequence and thus of presumably similar structure (see section D8). None of the chimeras in which a domain from one enzyme was attached to the corresponding partner in the other is active. The specific intrachain and interchain side-chain interactions also have to evolve for the Correcting packing.32... 

The first enzyme that was demonstrated to contain nickel was urease (urea amidohydrolase) from jack bean. It catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The protein has a multimeric structure with a relative molecular mass of 590,000 Da. Analysis indicated 12 nickel atoms/mol. Binding studies with the inhibitors indicated an equivalent weight per active site of 105,000, corresponding to 2 nickel atoms/active site. During removal of the metal by treatment with EDTA at pH 3.7, the optical absorption and enzymatic activity correlated with nickel content. This, combined with the sensitivity of the enzyme to the chelating agents acetohydroxamic acid and phos-phoramidate, indicates that nickel is essential to the activity of the enzyme (1). 

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