Biocatalyst active/catalytic sites

The first two strategies are based on chemical and structural modifications of the starting material (substrate engineering), whereas the last two are related to the nature of active catalytic site of the enzyme (biocatalyst engineering). 

The active or catalytic site of any biocatalyst represents the region of structure devoted to biocatalysis in any given biocatalyst. Catalytic sites are usually surprisingly small and frequently occupy a very small area/volume of the overall structure. During the hrst stage... 

Steady state kinetic measurements of initial rates are made under pseudo-irreversible Uni Uni kinetic conditions where all but one substrate are in substantial excess and no product is present. Furthermore, where a biocatalyst has more then one catalytic site, steady state analyses should be performed under conditions where only one of the sites is active at any one time, or else where the catalytic sites are at least able to operate completely independently of each other. In the latter case, measured values of kc t should then be divided through by the number of catalytic sites to provide a value per catalytic site, otherwise known as the turnover number per catalytic site. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

The idea of using organic metal complexes as catalysts for electrochemical reactions (Jasinski, 1964) can be traced back to the biocatalysts in which such complexes often are the catalytically active sites and which are distinguished by a high catalytic activity. This area has seen a strong development starting in the 1960s. 

Catalytic reduction of oxygen directly to water, while not as yet possible with traditional catalyst technology at neutral pH, is achieved with some biocatalysts, particularly by enzymes with multi-copper active sites such as the laccases, ceruloplasmins, ascorbate oxidase and bilirubin oxidases. The first report on the use of a biocatalyst... 

In carboxypeptidase A [52, 53], the active-site Zn(n) ion plays essential catalytic roles and the guanidinium of Arg-145 recognizes the carboxylate anion of the substrates, thus making the enzyme an exopeptidase. Important features of carboxypeptidase A reproduced by 11 include the essential catalytic action of a metal ion and participation of a guanidinium group in substrate recognition, so that this polymer biocatalyst hydrolyzes unactivated amides, and exhibits selectivity toward amide bonds adjacent to carboxylate groups in the substrate. 

The goal of diiron model chemistry is to develop small molecule systems that accurately reproduce spectroscopic, structural, and more ambitiously, reactivity aspects of driron metaUoproteins. Despite being structurally similar, diiron enzymes carry out a variety of catalytic processes see Iron Proteins with Dinuclear Active Sites)Advancements in the synthesis and characterization of small molecule mimics for nonheme diiron enzymes have been tremendous in the last decade. Biomimetic studies have been carried out in efforts to reproduce the structural and functional aspects of these biocatalysts. Although this has been a challenging endeavor, much information regarding the structural and mechanistic aspects of catalytic intermediates has been obtained. 

Enzymes, the catalysts of biological systems, are remarkable molecular devices that determine the patterns of chemical transformations. They also mediate the transformation of one form of energy into another. The most striking characteristics of enzymes are their catalytic power and specificity. Catalysis takes place at a particular site on the enzyme called the active site. Nearly all known enzymes are proteins. However, proteins do not have an absolute monopoly on catalysis the discovery of catalytically active RNA molecules provides compelling evidence that RNA was an early biocatalyst (Section 2.2.2). 

Semi-synthetic enzymes are produced by the reconstitution of apo-proteins with artificial active sites that yield novel catalytic functions [237]. For example, reconstitution of apo-myoglobin with Co(II)-protoporphyrin IX results in a novel biocatalyst that is capable of hydrogenating acetylene derivatives or evolving hydrogen [209, 238]. By the modification of the reconstitution of apo-proteins with artificial redox-active cofactors and the covalent attachment of photosensitizer units, photo-... 

Bacterial mercuric reductase is a unique metal-detoxification biocatalyst, reducing mercury(II) salts to the metal. The enzyme contains flavin adenine dinucleotide, a reducible active site disulfide (Cys 135, Cys i4o), and a C-terminal pair of cysteines (Cys 553, Cys 559). Mutagenesis studies have shown that all four cysteines are required for efficient mercury(II) reduction. Mercury Lm-EXAFS studies for mercury(II) bound to both the wild-type enzyme and a very low-activity C-terminal double-alanine mutant (Cys 135, Cys uo, Ala 553, Ala 559) suggest the formation of an Hg(Cys)2 complex in each case (39). The Hg—S distances obtained were 2.31 A and are consistent with the correlation of bond length with coordination number presented above. Thus, no evidence was obtained for coordination of mercury(II) by all four active-site cysteines in the wild-type mercuric reductase. However, these studies do not define the full extent of the catalytic mechanism for mercury(II) reduction, and it is possible that a three- or four-coordinate Hg(Cys) complex is a key intermediate in the process. 

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