Enzymes temperature dependence

Fermentation. The term fermentation arose from the misconception that black tea production is a microbial process (73). The conversion of green leaf to black tea was recognized as an oxidative process initiated by tea—enzyme catalysis circa 1901 (74). The process, which starts at the onset of maceration, is allowed to continue under ambient conditions. Leaf temperature is maintained at less than 25—30°C as lower (15—25°C) temperatures improve flavor (75). Temperature control and air diffusion are faciUtated by distributing macerated leaf in layers 5—8 cm deep on the factory floor, but more often on racked trays in a fermentation room maintained at a high rh and at the lowest feasible temperature. Depending on the nature of the leaf, the maceration techniques, the ambient temperature, and the style of tea desired, the fermentation time can vary from 45 min to 3 h. More highly controlled systems depend on the timed conveyance of macerated leaf on mesh belts for forced-air circulation. If the system is enclosed, humidity and temperature control are improved (76). 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Figure 3.14 Idealized van t Hoff plot of the temperature dependence of the affinity of a peptide inhibitor for die enzyme hdm2.

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

Pyrethroids have low oral toxicity to mammals, and in general their insect (topical) to mammal (oral) toxicity ratio is much higher than that of the other major classes of insecticides [25]. As the reason, at least the following mechanisms are conceivable (1) negative temperature dependence - differences in body temperature between insects and mammals makes the insect nerves much more sensitive, (2) metabolic rate - insects metabolize the insecticide more slowly than mammals, and the metabolizing enzyme systems are different, and (3) differences in body size - insects will have less chance to metabolize the insecticides before reaching the target site [26]. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

The books, however, cannot yet be closed. Although the flexibilities of the yeast enzyme at 25 °C and thermophilic enzyme at 65 °C are similar, and although both show unmistakeable evidence of tunneling, the nature of the tunneling process appears to be different. This is another instance in which the temperature dependences of the isotope effects generate a complex and ill-understood picture. 

Lewis et al. (entry 11 of Table 2) examined the temperature-dependence of isotope effects in the action of both the human enzyme and the soybean enzyme, by measuring the relative amounts of per-protio and per-deuterio-13-hydroperoxy-products by HLPC. The observed effects are, therefore, composed of primary, secondary, and perhaps remote isotope-effect contributions. Isotope effects on fecat/ M for both enzymes (determined by competition between labeled substrates) are increased by high total substrate concentration, an effect previously observed but stiU ill-understood. At 100 /rM substrate, the effects are roughly independent of temperature below about 15 °C, and are about 60 (H/D) for the human enzyme and 100 (H/D) for the soybean enzyme. Above 15 °C, the effects decline to about 50 for the human enzyme and about 60 for the soybean enzyme, perhaps because non-isotope-sensitive steps become more nearly rate-limiting (see Chart 4). 

Because both the passive fluctuations and the modulating vibrations can require thermal excitation, this model is capable of accounting for temperature-dependent isotope effects, including those traditionally described by the BeU model. Theoretical studies, which will be the topic of the second and third parts of this three-part series of articles, have not yet produced a consensus on the contribution of specific protein motions to enzyme catalysis. 

Kinetic complexity definition, 43 Klinman s approach, 46 Kinetic isotope effects, 28 for 2,4,6-collidine, 31 a-secondary, 35 and coupled motion, 35, 40 in enzyme-catalyzed reactions, 35 as indicators of quantum tunneling, 70 in multistep enzymatic reactions, 44-45 normal temperature dependence, 37 Northrop notation, 45 Northrop s method of calculation, 55 rule of geometric mean, 36 secondary effects and transition state, 37 semiclassical treatment for hydrogen transfer,... 

The conversions conducted in both steps are currently based on empirical relationships that are more or less robust. For example, the relationship between the chlorophyll and carbon content in an average phytoplankton cell is dependent on factors that influence cell metabolism, including nutrient arailability, temperature, and light. The temperature dependence of photosynthesis is associated with an enzyme-mediated step in the Calvin cycle (Figure 7.6a). 

Removal of calcium from HRP C has a significant effect not only on enzyme activity and thermal stability, but also on the environment of the heme group. The calcium-depleted enzyme has optical, EPR, and H NMR spectra that are different from those of the native enzyme (211). Temperature dependence studies indicate that the heme iron exists as a thermal admixture of high- and low-spin states. Kinetic measurements at pH 7 show that ki, the rate constant for compound I formation, is only reduced marginally from 1.6 0.1 x 10 to 1.4 x lO M s , whereas k, the rate constant for compound II reduction, is reduced from 8.1 1.6 x 10 to 3.6 x lO M s (reducing substrate p-aminobenzoic acid), 44% of its initial value (211). There can be little doubt that this is the main reason for the loss of enzyme activity on calcium removal. 

The magnetic interactions in the reduced porcine and bovine enzymes are considerably weaker than in the oxidized forms. The temperature dependences of the intensity of the EPR signals afford an estimate of -7 and -5.5 1 cm l for the J values of the reduced porcine and bovine enzymes, respectively (73,81). The large shifts observed in the NMR spectra of both enzymes also indicate that the coupling is weak from the temperature dependence of the isotropic shifts, J is estimated to be ca. -10 cm i for reduced uteroferrin (81). By analogy to the proposed structure for semimetHrNs, the reduced enzymes would have a hydroxo bridge. 

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