Substrates coenzymes

The concentrations of substrate, coenzyme, buffer, activators, pH, etc., should provide maximum enzyme activities. 

Controlled internal concentrations of substrates, coenzymes and metal ions. In particular,... 

Enzymes. The specificity of an enzyme for its substrate, coenzyme or competitive inhibitor provides the basis for many affinity chromatographic separations. Enzymes may be extracted and purified using insolubilized substrates, coenzyme or inhibitors. Less frequently, enzymes are used as the ligands. 

As we saw earlier in this chapter, substrates are the molecules which undergo chemical change as a result of enzyme activity. Many enzymes will only operate when in the presence of essential co-factors or coenzymes. The term coenzyme is not entirely appropriate as it implies that, like enzymes themselves, these compounds do not undergo chemical change. This is not true and more accurate terminology would be co-substrate. Coenzymes are always much smaller than the enzymes with which they operate and are not heat sensitive as are the proteins. 

Fig. 18. Covalent substrate-coenzyme adducts in flavin biochemistry...

Figure 26. AH-T/ iS plot for supramolecular interaction of enzyme with substrates ( ), coenzymes (o), and inhibitors (a).

Notice that each step in the overall sequence changes the electronic or steric characteristics of the complex in a way that facilitates the next step.246 This is an important principle that is applicable throughout enzymology For an enzyme to be an efficient catalyst each step must lead to a change that sets the stage for the next. These consecutive steps often require proton transfers, and each such transfer will influence the subsequent step in the sequence. Some steps also require alterations in the conformation of substrate, coenzyme, and enzyme. One of these is the transimination sequence (Eqs. 14-26,14-39). On the basis of the observed loss of circular dichroism in the external aldimine, Ivanov and Karpeisky suggested that a... 

The NADP-IDH from Escherichia coli has been thoroughly studied. It is a dimeric protein of two identical 40-kDa subunits. High-resolution X-ray crystal structures have been determined for the enzyme with and without substrate [16,17], and for the pseudo-Michaelis complex of the enzyme with isocitrate and NADP [18], Structures of sequential intermediates formed during the catalytic action of IDH are also available [19], Additionally, the kinetic and catalytic mechanisms have been determined in detail [20], Amino acid residues which are involved in interactions with substrate, coenzyme, metal ions, and catalysis have been identified [10,21],... 

Enzymes are highly selective of the substrates with which they interact and in the reactions that they catalyze. This selective nature of enzymes collectively known as enzyme specificity can be best illustrated with oxidoreductases (dehydrogenases), which display substrate and bond specificities (e.g., acting on —CHOH—, versus —CHO versus —CH—CH— versus —CHNH2, and cis versus trans for unsaturated substrates), coenzyme specificity (e.g., NAD(H) versus NADP(H)), chiral stereospecificity (d- versus l- or R- versus S-stereoisomers), and prochiral stereospecificity (A versus B corresponding to proR- versus proS isomers and re face versus si face, respectively). The table lists some dehydrogenases and their coenzyme, substrate, product and stereospecificities (You, 1982) ... 

Interaction with substrates, coenzymes, and allosteric effectors... 

The known coenzyme Bi2-dependent enzymes all perform chemical transformations in enzymatic radical reactions that are difficult to achieve by typical organic reactions. Homolytic cleavage of the Co bond of the protein-bound coenzyme B12 (3) to a 5 -deoxy-5 -adenosyl radical (9) and cob(n)alamin (5) is the entry to reversible H-abstraction reactions involving the 5 -position of the radical (9). Indeed, homolysis of the Co bond is the thermally most easily achieved transformation of coenzyme B12 (3) in neutral aqueous solution (with a homolytic (Co-C)-BDE of about 30 kcal mol ). However, to be relevant for the observed rates of catalysis by the coenzyme B12-dependent enzymes, the homolysis of the Co-C bond of the protein-bound coenzyme (3) needs to be accelerated by a factor of about 10 , in the presence of a substrate. Coenzyme B12 might then be considered, first of aU, to be a structurally sophisticated, reversible source for an alkyl radical, whose Co bond is labihzed in the protein-bound state (Figure 8), and the first major task of the... 

The redox states of the flavin cofactor in a purified flavoenzyme can be conveniently studied by optical spectroscopy (see also Elavoprotein Protocols article). Oxidized (yellow) flavin has characteristic absorption maxima around 375 and 450 nm (Fig. lb and Ic). The anionic (red) and neutral (blue) semiquinone show typical absorption maxima around 370 nm and 580 nm, respectively (Fig. lb and Ic). During two-electron reduction to the (anionic) hydroquinone state, the flavin turns pale, and the absorption at 450 nm almost completely disappears (Fig. lb and Ic). The optical properties of the flavin can be influenced through the binding of ligands (substrates, coenzymes, inhibitors) or the interaction with certain amino acid residues. In many cases, these interactions result in so-called charge-transfer complexes that give the protein a peculiar color. 

A crude cell-free extract contained 20 mg of protein per milliliter. Ten microliters of this extract in a standard total reaction volume of 0.5 ml catalyzed the formation of 30 nmoles of product in 1 min under optimum assay conditions (optimum pH and ionic strength, saturating concentrations of all substrates, coenzymes, activators, and the like), (a) Express v in terms of nmoles/assay, nmoles x mT x min", nmoles X liter" x min", moles x liter" X min", M X min", (b) What would v be if the same 10 p,l of extract... 

There are reciprocal relationships between the parameters summarized above. On the one hand enzyme stability measurements strongly depend on the concentrations of substrates, coenzymes, buffers etc. in the assay. On the other hand the choice of an appropriate concentration level is a consequence of the enzyme kinetics investigated afterwards. A compromise has to be found between different optimization criteria e. g. a lower temperature leads to a reduced enzyme activity but results in a higher enzyme stability. In the example of the oxynitrilase reaction (Eq. (12)) a low pH value is a prerequisite for high enantiomeric purity of the product but lowers enzyme activity. As a consequence, only a rough optimization can be carried out at this level. 

Fig. 11. Schematic representation of the interactions between the substrate, coenzyme, and the active site residues in horse liver alcohol dehydrogenase. Not shown are the interactions between Arg-47 and the pyrophosphate backbone, and Asp-49, which forms a salt bridge with His-57, another ligand of the zinc atom. Because of the close proximity to residues having obvious catalytically important functions, alterations in the interactions between the coenzyme and Ser-48 and His-51 that are anticipated from the binding of acyclo-NAD could readily cause the observed changes in substrate specificity. Based on Ref. 38.

On the chemical level, enzyme-catalyzed recycling of substrates/coenzymes to improve sensitivity of an assay has been successfully demonstrated [27, 38, 39]. The principle is illustrated in the reaction sequence shown in figure 22.5. 

The complete name for lactate dehydrogenase is lactate NAD oxidoreductase. This systematic name tells us the substrate, coenzyme, and type of reaction catalyzed. 

Above we have used concepts and drawn detailed structures for various species involved in pyridoxal-P-dependent enzymes without presenting experimental evidence. This section gives an account of spectroscopic and chemical studies which have formulated our views on the structure of the binary (coenzyme-enzyme) and ternary (substrate-coenzyme-enzyme) complexes and the nature of chemical events which occur within them. 

The binary complex can exist as an equilibrium mixture of a number of species as shown in Fig. 43. The major species apparent in a number of binary complexes, e.g. glutamate.decarboxylase and aspartate aminotransferase [31,90] appear to be those absorbing at 420 and 333 nm which are attributed to structures 2 and 3 (Fig. 43) respectively as expected the ratio of these species is pH dependent. On the other hand, the ternary complex may exist as an equilibrium mixture composed of species from both binary and ternary complexes, thus producing a composite electronic absorption profile however, the ternary complex of aspartate aminotransferase exhibits only two major absorption maxima at 430 and 340 nm due to substrate-coenzyme Schiff base and enzyme bound pyridoxamine-P respectively [90]. It is interesting to note the spectra observed for aspartate aminotransferase... 

Structure and stereochemistry of the substrate-coenzyme bond in ternary complexes... 

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