Factors that change the activity of an enzyme

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

Unfortunately, the term coenzyme is sometimes confused with cofactor , but the difference is fundamentally important. Coenzymes do not function as part of the enzyme. They are, in fact, substrates (or products) in the reaction. The term coenzyme is usually used to describe 

An enzyme in a biochemical pathway may have two substrates, one of which is a cofactor. The other which is usually the substrate involved in the primary flax through the pathway is termed a pathway-substrate in this book. 

Many factors can change the catalytic activity of an enzyme. Those discussed in this book are the concentrations of substrates, protons (pH), temperature and inhibitors. The effects of these are discussed from a qualitative point of view, with more quantitative descriptions presented in Appendices 3.3, 3.4 and 3.5. 

The effects of these factors and the means by which they are studied are usually described as enzyme kinetics. The basic information required to study enzyme kinetics is as follows. 

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An important factor related to the activity of an immobilized enzyme is the chemical environment immediately surrounding the protein. In the previous section, we discussed the infusion of a free probe into an immobilization matrix to assay the relative hydrophobicity of the immobilization matrix. In general, it is useful to confirm that modification of the polymer prior to its formation into an immobilization matrix introduced no essential change in chemistry (i.e., did not make the matrix relatively more hydrophobic or hydrophilic). What the technique does not do, however, is definitively probe the chemical microenvironment immediately surrounding the enzyme. 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

There is evidence that poly(ADP-ribosyl)ation, in response to DNA damage (review [1]), permits changes in chromatin structure [2] which may facilitate the activity of repair enzymes [3] and become a major factor in the control of cell-cycle traverse [4], This paper explores the role of chromatin structure and poly(ADP-ribosyl)ation (using the inhibitor, 3-aminobenzamide 3AB [5]) in the responses of human cells to either a DNA specific ligand (Hoechst dye 33341 Ho33342) or X-radiation. Cells derived from an ataxia telangiectasia (A-T) patient have been included in the study to provide a h) ersensitive control (reviews [6, 7]) in which anomalous cell survival and cell-cycle responses [8-10] to radiation may reflect a primary defect in chromatin structure [11,12]. 

Although the Arg Gin point mutation did not generate an enzyme with true acyl hydrolase activity, the ability of the domain to allow transfer of an acyl chain to the active site serine is different from the activity exhibited by WT PedD, which is unable to catalyse either transfer or hydrolysis of an acyl chain (Fig. 5.18). These results indicate that the point mutation has changed the activity of PedD towards that of PedC, with the factors dictating the ability catalyse hydrolysis yet to be identified. 

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