Several thermophilic enzymes show indeed a remarkable stability at high temperatures, while others are unstable in pure preparations and obviously need the stabilizing capacity of cellular components. Tests at high temperatures are more complicated, not only due to more difficult thermostatting. Other components of the

assay mixture may become unstable and oxidation processes are accelerated. Besides the enzyme itself, substrates, co-substrates and cofactors5 are ATR inhibitor the most important components of the enzyme assay. Their state, their purity and stability is of particular importance and highest demands have to be made for these substances. With respect to the substrates a significant aspect must be considered. Usually it is taken that the enzyme has a defined substrate according to its physiological function, www.selleckchem.com/products/ABT-263.html as lactate dehydrogenase oxidizing lactate to pyruvate, or fumarase forming malate from fumarate. However, the substrate is not clearly defined in every case. Many enzymes show broad specificity, accepting also substances structurally related to the physiological substrate, like alcohol dehydrogenase, which reacts with various alcohols. The same holds for cofactors. Divalent cations are essential cofactors for many catalytic reactions

and they can often be substituted by other divalent cations. An interesting example is glucose isomerase, a microbial enzyme. Its physiological substrate is xylose, which becomes isomerized to xylulose with Mn2+ acting as essential cofactor. Due to its capacity to isomerize also glucose to the more valuable sugar fructose, the enzyme gained great interest in biotechnology. This non-physiological reaction proceeds more efficient with Co2+ than with Mn2+. So the change of the substrate causes also a change of the cofactor (Antrim et al., 1979 and Lehmacher and Bisswanger, 1990). In other cases the physiological substrate IMP dehydrogenase is replaced by an artificial, synthetic substrate, e.g. if the physiological substrate is unstable or, as in the case

of proteases, if the (protein) substrate is not well defined, rather the single peptide bond within the protein must be regarded as the genuine substrate. If the enzyme accepts different substrates, the question arises which substrate should be used for the enzyme assay? Due to the varying catalytic efficiency, results obtained for the same enzyme, but with different substrates, will hardly be comparable. The efficiency of a substrate is determined by its Km value, the lower this value the better the substrate. Usually the most efficient substrate may be taken, but also other aspects must be considered, like the availability, stability, solubility and the accessibility to a detection method. Sometimes natural substrates are modified to facilitate the detection. So it is not always the physiological substrate which is applied for the enzyme assay, but it is obvious that for comparison of the results the same substrate must always be used.