Active Site Chemistry

Most quantum chemical modelling studies deal with active site chemistry. That is, the calculations do not really focus on how substrates and products get to and from the active site. Rather, they concentrate on the sequence of events following the arrival of substrate in the active site pocket and seek to uncover the mechanism of conversion of substrate to one or more intermediates and/or product. The obvious reason for such an approach is the assumption that the vast bulk of the protein molecule can be ignored but raises the thorny issue of whether this is a valid assumption. In practical terms, it is not possible (and arguably not desirable) to treat the entire protein quantum mechanically. Moreover, since one of the main roles of the protein is substrate selection and delivery to the active site, and since the computer modeller has explicit control over this feature, one might conclude that there is no need to consider the bulk of the protein molecule. However, the protein backbone may exert a structural influence on the active site—the entatic state [34]—while the groups around the active site produce an electrostatic field different from the in vacuo state which is the default domain of quantum chemistry. In summary, it is obviously critically important to develop a reasonable chemical model of the active site if any conclusions drawn from the calculations are to be believed. 

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This nomenclature scheme, based on active site chemistry, is complimentary to the endo/exo nomenclature described above. Thus, the two schemes are frequently combined. For example, trypsin (a digestive enzyme) is often descriptively referred to as a serine endopepti-dase. 

As the focus of this review is on copper-dioxygen chemistry, we shall briefly summarize major aspects of the active site chemistry of those proteins involved in 02 processing. The active site structure and chemistry of hemocyanin (He, 02 carrier) and tyrosinase (Tyr, monooxygenase) will be emphasized, since the chemical studies described herein are most relevant to their function. The major classes of these proteins and their origins, primary functions, and leading references are provided in Table 1. Other classes of copper proteins not included here are blue electron carriers [13], copper-thiolate proteins (metallothioneines) [17], and NO reductases (e.g., nitrite [NIR] [18] or nitrous oxide [19]). 

As observed in other systems, the obvious difficulty in elucidating reaction mechanisms based on static structural snapshots subsequently initiated structural-dynamic theoretical studies of metalloproteinases. The active site chemistry of zinc-dependent enzymes has been studied using a variety of theoretical approaches. For example, mixed quantum mechaiucal/molecular calculations and classical molecular dynamic simulations have been employed, especially studies using density functional methods on redox-active metal centers (42). 

The first crystal structure information on a blue copper protein, for poplar plastocyanin in the Cu(II) state, was published in 1978 (2, 3). Since then, the Cu(I) state and related apo and Hg(II) substituted forms (5, 6), the green algal plastocyanin from Enteromorpha prolifera [Cu(II)] (7), azurin from Alcaligenes denitrificans [Cu(II) and Cu(D] (8, 9), azurin from Pseudomonas aeruginosa [Cu(II)] (10, 11), as well as pseudoazurin from Alcaligenes faecalis S-6 (12), and the cucumber basic protein, both in the Cu(II) state, have been published (13), making this one of the best-documented class of proteins. In addition, information as to three-dimensional structure in solution has been obtained from two-dimensional NMR studies on French bean and Scenedesmus obliquus plastocyanins (14,15). This review is concerned in the main with the active site chemistry. Other recent reviews are listed (16-20). 

A number of techniques have contributed to the present understanding of the active site chemistry of blue copper proteins. Perhaps foremost is that involving X-ray crystallography. A wide range of physical... 

Increasing the substrate range of an enzyme could be thought of as improving a promiscuous activity. However, here we will use a more strict definition in which a promiscuous activity involves catalysis of a reaction with a different class of substrate. Many enzymes are promiscuous in the sense that they can catalyze other reactions. This is not surprising considering that the enormous variety of enzymes that exist utilize only a small number of active site chemistries and structural scaffolds. Thus, an almost identical enzyme could catalyze lactone hydrolysis or phosphotriester hydrolysis. Because these activities are often very weak to begin with, directed molecular evolution experiments to improve these activities often result in remarkable improvements. 

Lui, S.M. and J.A. Cowan (1994). Direct reversible protein electrochemistry at a pyrol54ic graphite electrode. Characterization of the redox thermodynamics of the Fe4S4-siroheme prosthetic center in the hexameric dissimilatory sulfite reductase and the monomeric assimilatory snlflte reductase from desulfovibrio vulgaris (Hilden-hourgh). Systematic pH titration experiments and implications for active site chemistry./.Am. Chem. Soc. 116, 11538-11549. 

Garindarajai, K., Unni Nair, Ramasami, N and Ramaswamy, D. (1987) "Active Site Chemistry of Lysyl Oxidase" J. Inorg. Biochem., 29 111-18. 

These simple examples illustrate that many of the basic active site chemistry of enzymes can be reproduced with simple organic models in the absence of proteins. The role of the latter is of substrate recognition and orientation and the chemistry is often carried out by cofactors (coenzymes) which also have to be specifically recognized by the protein or enzyme. The last chapter of this book is devoted to the chemistry of coenzyme function and design. 

Various studies indicate the participation of at least three amino acid residues in the active site chemistry of ribonuclease two histidines and one lysine. RNA hydrolysis (Fig. 3.6) proceeds by two steps transesterification followed by hydrolysis. Note that at physiological pH, one of the two imidazole rings is protonated while the other is not. The imidazole rings... 

A number of other enzymes which catalyze the hydrolysis of phosphoesters are of biological importance. These include cyclic purine phosphodiesterase (little is known about its active site chemistry at present, but more shall be said about its biological role shortly) and the phosphatases. Acid and alkaline phosphatase catalyze the hydrolysis of phosphomonoesters to the corresponding alcohol and inorganic phosphate. Their pH optimums are 5.0 and 8.0, respectively hence their names. Both form covalent enzyme-substrate intermediates ... 

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