Dehydrogenase Active Site Models

it was observed that the reaction barriers for the ratedetermining steps were well above the general kinetic requirements of an enzymatic catalytic process (15-20 kcal/mol). Then, the three water molecules (W362, W615, W213) in the catalysis area were also taken into account by adding them to the contents of Model B so that the Ca first-shell coordination sphere is complete and tested upon methanol oxidation in protein environment to provide extended analysis on the oxidation mechanisms. 

MDH active site models were geometry minimized at the BLYP/DNP level with no constrains, and further tested upon methanol oxidation as explained in the following Section. The reaction mechanisms are tested in gas-phase only for Model A, since the inclusion of solvation effects (for protein environment) generally creates a pronounced effect on the calculated energetics for this model as observed from the literature because of the incomplete representation of the first-coordination shell of Ca. Model B, which considers the complete coordination sphere of the ion, was used for testing the mechanism with and without the pres- 

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Exploitation of the complementary specificities of enzymes from different sources towards the same racemic substrate permits very precise control of the product stereochemistry. For example, any one of the three diastereomeric 2-decalols (94)-(96) can be obtained at will from ( )-tra/iJ-2-decalone (81 R = H) using the alcohol dehydrogenases HLADH, MJADH or CFADH, respectively (Scheme 40). The stereospecificities of these three enzymes are well documented and a simple active site model of predictive value is available for each. 55 Racemic bridged bicyclic ketones are similarly discriminated, either... 

Figure 6.14 Active site models for xanthine oxidase and nicotinic acid dehydrogenase and their atom transfer reactivity.

Gourlay C, Nielsen DJ, White JM, Knottenbelt SZ, Kirk ML, Young CG (2006) Paramagnetic active site models for the molybdenum — copper carbon monoxide dehydrogenase. J Am Chem Soc 128(7) 2164-2165. doi 10.1021/ja056500f... 

E. Nordling, H. Jomvall, B. Persson, Medium-chain dehydrogenases/reductases (MDR). Family characterizations including genome comparisons and active site modelling, Eur. J. Bio-chem. 269 (2002) 4267-4276. 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

Ribas dePoplana L, Fothergill-Gilmore LA. The active site architecture of a short chain dehydrogenase defined by site-directed mutagenesis and structure modeling. Biochemistry 33 1994 7047-7055. 

The NAD+-dependent alcohol dehydrogenase from horse liver contains one catalytically essential zinc ion at each of its two active sites. An essential feature of the enzymic catalysis appears to involve direct coordination of the enzyme-bound zinc by the carbonyl and hydroxyl groups of the aldehyde and alcohol substrates. Polarization of the carbonyl group by the metal ion should assist nucleophilic attack by hydride ion. A number of studies have confirmed this view. Zinc(II) catalyzes the reduction of l,10-phenanthroline-2-carbaldehyde by lV-propyl-l,4-dihy-dronicotinamide in acetonitrile,526 and provides an interesting model reaction for alcohol dehydrogenase (Scheme 45). The model reaction proceeds by direct hydrogen transfer and is absolutely dependent on the presence of zinc(II). The zinc(II) ion also catalyzes the reduction of 2- and 4-pyridinecarbaldehyde by Et4N BH4-.526 The zinc complex of the 2-aldehyde is reduced at least 7 x 105 times faster than the free aldehyde, whereas the zinc complex of the 4-aldehyde is reduced only 102 times faster than the free aldehyde. A direct interaction of zinc(II) with the carbonyl function is clearly required for marked catalytic effects to be observed. 

Figure 3-24. A zinc(ii) complex which acts as a functional model for the hydride transfer reaction which occurs at the active site of the enzyme liver alcohol dehydrogenase.

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Figure 1. (a) View of the inside of the Methanol Dehydrogenase (MDH) enzyme with the active site in stick model. The solid surface represents the solvent-accessible MDH external surface showing the binding pocket, (b) View from the binding pocket of the entire MDH active site. Amino acids labels denote their location in the sequence obtained from the entry 1W6S (Methylobacterium Extorquens W3A1 ) of the Protein Data Bank. 

The substrate models concerned were fitted into the model of the active site of alcohol dehydrogenase-nicotinamide adenine dinucleotide (ADH-NAD) with VDW contacts, etc. not considered explicitly. 

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