Enzyme-substrate complexes, crystals

From these observations, we have noticed the similarity of the simple lattice inclusions to the more sophisticated assemblies of molecules (e.g. cyclodextrins 76 and proteins 78 where the formation of H-bonded loops was first detected and described. Conclusively the motive for the formation of simple inclusion crystals and of more complex associates between high and low molecular weight compounds, such as enzyme-substrate complexes, can be traced back to the same source. 

The enzyme carboxypeptidase A is particularly amenable to structural investigation crystal structures of the enzyme, of complexes of the enzyme with substrates, substrate analogues and inhibitors, and of transition-state analogues are available. To isolate an enzyme-substrate complex for a one-substrate enzyme reaction, or for an enzyme reaction where water is a... 

These results suggest that the crystallographic determination of the structure of a productive enzyme-substrate complex is feasible for lysozyme and oligosaccharide substrates. They also provide the information of pH, temperature, and solvent effects on activity which are necessary to choose the best conditions for crystal structure work. The system of choice for human lysozyme is mixed aqueous-organic solvents at -25°C, pH 4.7. Data gathered on the dielectric constant, viscosity, and pH behavior of mixed solvents (Douzou, 1974) enable these conditions to be achieved with precision. 

Tables VIII-XI show examples of pon variations of several buffers. With such tables, it is easy to adjust any desired pan value in mixed solvents at any selected temperature or in a given range of temperatures. We will see in Section III,E how these values are essential to investigate safely both crystal structure and productive enzyme-substrate complexes in the crystalline state.

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

An important advantage of the inclusion complexes of the cyclodextrins over those of other host compounds, particularly in regard to their use as models of enzyme-substrate complexes, is their ability to be formed in aqueous solution. In the case of clathrates, gas hydrates, and the inclusion complexes of such hosts as urea and deoxycholic acid, the cavity in which the guest molecule is situated is formed by the crystal lattice of the host. Thus, these inclusion complexes disintegrate when the crystal is dissolved. The cavity of the cyclodextrins, however, is a property of the size and shape of the molecule and hence it persists in solution. In fact, there is evidence that suggests that the ability of the cyclodextrins to form inclusion complexes is dependent on the presence of water. Once an inclusion complex has formed in solution, it can be crystallized however, in the solid state, additional cavities appear in the lattice, as in the case of the hosts previously mentioned, which enable the inclusion of further guest molecules. ... 

The enzymes of this type that have been characterized contain some type of redox-active cofactor, such as a flavin (3), or a metal ion (heme iron, non-heme iron, or copper), or both (4-6). Our understanding of the mechanism of these enzymes is most advanced in the case of the heme-containing enzyme cytochrome P450. But in spite of the availability of a crystal structure of an enzyme-substrate complex (7) and extensive information about related reactions of low molecular weight synthetic analogues of cytochrome P450 (8), a detailed picture of the molecular events that are referred to as "dioxygen activation" continues to elude us. 

The currently accepted chemical mechanism for the reaction of RNase A was deduced by an inspired piece of chemical intuition before the crystal structure was solved.194 It was found that the pH-activity curve is bell-shaped, with optimal rates around neutrality. The pH dependence of kQJKM shows that the rate depends upon the ionization of a base of pKa 5.22 and an acid of pATfl 6.78 in the free enzyme, whereas the pH dependence of kcax shows that these are perturbed to pKa values of 6.3 and 8.1 in the enzyme-substrate complex. It was proposed that the reaction is catalyzed by concerted general-acid-general-base catalysis by two histidine residues, later identified as His-12 and His-119 (reactions 16.36 and 16.37). 

Spectroscopic studies of the enzyme-substrate complex show that the 8-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) thiolate coordinates to the metal center. First, there is a decrease in the Mossbauer isomer shift of the Fe(II) center from 1.2 to 1.0 mm/sec, indicating a more covalent Fe(II)-ligand environment [195], Second, an intense band appears at 390 nm in the visible spectrum of Cu(II)IPNS upon addition of ACV, which is associated with a thiolate-to-Cu(II) charge transfer transition found for tetragonal copper(II) centers [196], Last, EX-AFS analysis of the Fe(II)IPNS-ACV complex indicates the presence of a sulfur scatterer at ca. 2.3 A, which is a distance typical of Fe(II)-thiolate coordination [197,198], The very recently elucidated crystal structure of the Fe(II)IPNS-ACV complex confirms the thiolate coordination [199],... 

Figure 20 Crystal structure of the enzyme-substrate complex of naphthalene dioxygenase Fe-NDO-napthalene (lOVG.pdb)...

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