Bovine enzyme-substrate interactions

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

The role of the Zn atom in E. coU alkaline phosphatase, which catalyzes the phosphorylation of serine 99 in the amino add sequence to form the covalent enzyme-phosphate intermediate is well understood. Unfortunately, knowledge of the molecular mechanisms underlying the enzymic activity of the purple add phosphatases is far more rudimentary. Spectroscopic studies utilizing inhibitors and perturbants (Sect. III.B.10), such as phosphate and molybdate, indicate that substrate binds close to the binuclear iron cluster of uteroferrin and bovine spleen phosphatase Most likely, the substrate interacts with the redox active iron of the pair . ... 

Bovine a-lactalbumin is one of the two enzymes in lactose synthetase, and its amino acid sequence shows striking similarities to that of lysozyme.118 A model based on the lysozyme model has been built, and the side-chain interactions found are convincing, showing that the model is essentially correct. The active cleft in the crystal is, however, shorter than that in the model, and is consistent with a mono- or di-saccharide as the substrate. Thus, the lysozyme structure may serve as a model for some enzymes that synthesize and hydrolyze carbohydrates. 

A plot of log Kj against log 1/kj (Figure 5) clearly reveals an excellent linear relationship (r-0.97) indicating that these N-methoxy N-methylcarbamates may in fact act as TSA, and the corresponding N-methylcarbamates are poor substrates and part of their inhibitory potency is likely to be due to their resemblance to the TS complex in the reaction of the enzyme with its carboxy ester substrate. Additional supportive evidence is that when Wustner et al. (41) tried to prove that the ring substituents in both series of compounds interact similarly with the bovine erythrocyte AChE active site they found a disappointing correlation between Kj and Kj and a better correlation between Ki and kj respectively for N-methoxy N-methyl and N-methylcarbamates. 

Enzyme Models .—Two general mechanisms have been proposed for hydrolytic reactions catalysed by bovine pancreatic carboxypeptidase A the first involves formation of an anhydride intermediate and the second involves the residue Glu-270 as a general base. Work on model systems and the enzyme indicates that the general base mechanism is the correct one and a consistent mechanism (Scheme 4) has been proposed in which both zinc and Arg-145 interact with the substrate. 

Many enzymes or other proteins are stabilized by interaction with small ligands (substrates, cofactors, inhibitors, products). Nonspecific protection by macromolecules and stabilization by specific protein—protein interaction are generally less readily quantified. As a model system in which to determine effects of protein—protein interaction on conformational (thermal) stability, Donovan and Beardslee (1975) selected the proteinase—inhibitor association between bovine 3-trypsin and STI or chicken ovomucoid. They observed that both protein components were stabilized in the 1 1 complexes and, furthermore, that each trypsin—inhibitor complex showed one endotherm in the DSC. No stabilization was observed for a weaker electrostatic complex between ovalbumin andjysozyme. They interpreted the stabilization as an increase in kinetic thermal stability, i.e., a decrease in the rate of denaturation at a given temperature. Results obtained by other methods (Edelhoch and Steiner, 1965 Laskowski and Sealock, 1971 Jibson et al., 1981) show that proteinase—inhibitor association generally involves no major conformational changes in the proteins. 

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