Active site structure determination

Structural studies on substrates and intermediates at the active site. Structural determination of the various reactive complexes at the active site of RNase, utilizing X-ray and computational analysis, has reached such a level that Fersht (1984) was able to portray the events involved in the RNase reaction in a series of freeze-frame photographs . Two studies approaching the mechanism of the cyclization step of RNase through theoretical calculations are of particular note. 

Ligand bound to enzyme active site, structure determined by joint X-ray and neutron diffraction. 

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

Figure 5. Active site structure of the met form of the E. coli R2 protein of ribonucleotide reductase as determined in a 2.2-A resolution X-ray crystallographic study (14, 102).

Figure 6. The active site structure of the catalytically inactive form of GO (pH 4.5 acetate buffer) as determined in a 1.7-A resolution X-ray crystallographic study (119, 120).

Dawson, J. H. and Sono, M. (1987) Cytochrome P450 chloroperoxidase thiolate-ligand heme enzymes. Spectroscopic determination of their active site structure and mechanistic implication of thiolate ligation. Chem. Rev. 87, 1255-1276. 

Figure 15. Active-site structure of (Na + K )-ATPase as determined by 1H, 205Tl, 3IP, and 7Li NMR, Mn3 EPR, and kinetic studies...

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

Dawson JF1, Sono M (1987) Cytochrome P-450 and chloroperoxidase - thiolate-ligated heme enzymes - spectroscopic determination of their active-site structures and mechanistic implications of thiolate ligation. Chem Rev 87 1255-1276... 

D of protoporphyrin-IX depends on the steric constraints of the substrate binding pocket. Ortiz de Montellano (59) has used this selectivity to probe the active site structure of several heme enzymes. The structure of phenyl-cyt P-450cam has been determined by X-ray crystallography and indicates that N-phenyl heme formation is an accurate, low-resolution probe of active site structure. 

As many organic compounds may transform simultaneously through mono molecular (intramolecular) and bimolecular (intermolecular) processes, it is easy to understand that the shape and size of the space available near the active sites often determine the selectivity of their transformation. Indeed the transition state of a bimolecular reaction is always bulkier than that of a monomolecular reaction, hence the first type of reaction will be much more sensitive to steric constraints than the second one. This explains the key role played by the pore structure of zeolites on the selectivity of many reactions. A typical example is the selective isomerization of xylenes over HMFI the intermediates leading to disproportionation, the main secondary reaction over non-spatioselective catalysts, cannot be accommodated at its channel intersections (32). Furthermore, if a reaction can occur through mono and bimolecular mechanisms, the significance of the bimolecular path will decrease with the size of the space available near the active sites (41). 

Further, the active sites co-exist with the rest of the surface and bulk of the heterogeneous catalyst, and there is keen interest in determining the relationship between the bulk, surface, and active site structure in any given catalytic system. 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

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