Enzymes, reactions with Subject

These were relatively low-resolution structures, and with refinement some errors in the initial structural assignments have been detected (4-7). Since the structures were first reported the subject has been extensively reviewed in this series (8) and elsewhere 9-15). This review will focus on the structure, biosynthesis, and function of the met-allosulfur clusters found in nitrogenases. This will require a broader overview of some functional aspects, particularly the involvement of MgATP in the enzymic reaction, and also some reference will be made to the extensive literature (9, 15) on biomimetic chemistry that has helped to illuminate possible modes of nitrogenase function, although a detailed review of this chemistry will not be attempted here. This review cannot be fully comprehensive in the space available, but concentrates on recent advances and attempts to describe the current level of our understanding. 

The slowing down of enzyme reactions has often been attributed to reaction with, or equilibrium between, the enzyme and its substrate or between the enzyme and the products of its action. In order to determine the influence of the products of the action of pancreatic amylase on the extent of the hydrolysis of starch, portions of its hydrolysis mixtures were subjected to efficient dialysis during hydrolysis and the results compared with aliquots of the reaction mixture which had been treated in the same way except for dialysis.41 The results of such experiments... 

Many of the specific enzymes involved with phase I reactions are subject to induction (see Section 3.3.3), that is increased synthesis by activation of the particular genes. Two clinical consequences arise from this ... 

The existence of various isoforms of many of the enzymes involved with phase I and phase II reactions has been noted above. Key enzymes are also subject to variation by genetic polymorphisms so there may be considerable difference in the metabolic efficiency between individuals and between different ethnic groups. Such genetic differences account for most the variability we see between individuals capacity to metabolise certain drugs. For example, refer to Section 6.4.3. 

Intramolecular reactions often differ from their intermolecular counterparts in the exceptionally high rates that are observed and some reactions can occur intramolecularly that are impossible between separate molecules. Because of the importance of intramolecular catalysis, the subject has been reviewed frequently, particularly with reference to its connection with enzymic catalysis (Page, 1973, 1984 Fife, 1975 Jencks, 1975 Kirby, 1980 Fersht, 1985 Menger, 1985). The present coverage is limited to examples of intramolecular catalysis that owe some of their efficiency to intramolecular hydrogen bonding. The role that hydrogen bonds play in enzymic reactions is discussed in Section 5. 

Imbalance in the stoichiometry of polycondensation reactions of AA-BB-type monomers can be overcome by changing to heterofunctional AB-type monomers. Indeed, IIMU has been subjected to bulk polycondensation using lipases as catalyst in the presence of 4 A molecular sieves. At 70 °C, CALB showed 84% monomer conversion and a low molecular weight polymer (Mn 1.1 kDa, PDI 1.9). No significant polymerization was observed with other lipases (except R cepacia lipase, 47% conversion, oligomers only) and in reference reactions with thermally deactivated CALB or in the absence of enzyme. Further optimization of the reaction conditions (60wt% CALB, II0°C, 3 days, 4 A molecular sieves) gave a polymer with Mn of 14.8 kDa (PDI 2.3) in 86% yield after precipitation [42]. 

An unusual photochemical reaction of 2-pyridones, 2-aminopyridinium salts and pyran-2-ones is photodimerization to give the so-called butterfly dimers. These transformations are outlined in equations (13) and (14). Photodimerization by [2+2] cyclization is also a common and important reaction with these compounds. It has been the subject of particular study in pyrimidines, especially thymine, as irradiation of nucleic acids at ca. 260 nm effects photodimerization (e.g. equation 15) this in turn changes the regular hydrogen bonding pattern between bases on two chains and hence part of the double helix structure is disrupted. The dimerization is reversed if the DNA binds to an enzyme and this enzyme-DNA complex is irradiated at 300-500 nm. Many other examples of [2+2] photodimerization are known and it has recently been shown that 1,4-dithiin behaves similarly (equation 16) (82TL2651). 

The mechanisms behind lipid oxidation of foods has been the subject of many research projects. One reaction in particular, autoxida-tion, is consistently believed to be the major source of lipid oxidation in foods (Fennema, 1993). Autoxidation involves self-catalytic reactions with molecular oxygen in which free radicals are formed from unsaturated fatty acids (initiation), followed by reaction with oxygen to form peroxy radicals (propagation), and terminated by reactions with other unsaturated molecules to form hydroperoxides (termination O Connor and O Brien, 1994). Additionally, enzymes inherent in the food system can contribute to lipid oxidization. 

To 17 parts by volume of the crude enzyme solution are added 5 parts of kanamycin B, 50 parts by volume of 0.5 M phosphate buffer (pH 7.0), 100 parts by volume of 1 M adenosine triphosphate solution, 50 parts by volume of 0.1 M magnesium acetate solution and 50 parts by volume of 0.1 M 2-mercaptoethanol, which is filled up to 500 parts by volume with distilled water. The mixture is subjected to enzymic reaction at 37°C for 20 h. 

Immobilization of enzymes. Enzymes consist of amino acids which contain reactive groups such as amino(lysine, c-terminus), thiol (cysteine), carbonyl (aspartate, glutamate and c-terminus), aromatic hydroxyl (tyrosine) and aliphatic hydroxyl (serine and threonine). Chemical, ionic or chelation reactions with such groups can enable us to attach the amino acids and hence proteins to insoluble, inert supports. Immobilization is one of the best ways of stabilizing enzymes. There is a vast literature on this subject and the reader is directed to Barker (9) and Goughian et al (10) for further reading on specific systems, techniques and applications of immobilization. 

Enzymes that are subject to control signals generally fulfill two criteria they are present at low enzymatic activities and catalyze reactions that are not at equilibrium under cellular conditions. Both criteria arise because control enzymes are likely to be those catalyzing the slowest (rate-determining) step in a metabolic pathway. This is likely to be the case if an enzyme is present at low activity. If this is the case, the enzyme-catalyzed reaction is unlikely to be at equilibrium in vivo because there is insufficient enzyme present to allow equilibration of its reactants before they react with other compounds. 

Being a protein, an enzyme can lose its catalytic properties when subjected to agents such as heat, strong acids or bases, organic solvents, or other materials that denature the protein. Each enzyme catalyzes a specific reaction or a group of reactions with certain... 

Reductions of carbonyl groups with lithium aluminium hydride or sodium borohydride occur by hydride transfer to carbon from aluminium or boron, respectively. The course of reaction is subject to steric approach control and product development control [43-45]. Enzymic reactions may or may not form the epimer favoured in the chemical reduction. This has been discussed elsewhere [46]. It is quite clear that the steric course of a dehydrogenase reaction is determined by the structure of the enzyme. 

A brief report has appeared in which a wild-type esterase from Pseudomonas fluorescens (PFE), which shows no activity in the hydrolysis of the ester 16 (Fig. 11.21), was subjected to mutagenesis using the mutator strain Epicurian coli XL 1-Red [83], This resulted in a variant which catalyzes the reaction with an ee of 25 %. The absolute configuration of the major product ((R)- or (S)-17) was not determined. Sequencing of the esterase-variant revealed that two point mutations, A 209D and L181V, had occurred. Since the structure of the enzyme is unknown, a detailed interpretation was not possible, although reasonable speculations were made. 

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