Crystalline enzyme-substrate complex

G. Strategies for Trapping Crystalline Enzyme-Substrate Complexes. 320... 

Much of the recent crystallographic work on IPNS has involved brief exposure of crystalline enzyme-substrate complexes to hyperbaric O2 followed by cryo-... 

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. 

Phosphine complexes are generally stable, non-ionic, soluble in organic solvents, and obtainable as pure highly crystalline (often highly coloured) compounds. Some of these compounds have important catalytic properties, which can be compared with the action of metal/enzyme/substrate complexes in which the bonding is of the type M-O-P (Chapter 11.4). 

In addition to x-ray elucidation of the structure of the crystalline enzyme, the structure of a crystalline complex of lysozyme and tri(N-acetylglucosamine) was determined (Phillips, 1966). The trisaccharide occupied sites A, B, and C. Assuming that binding of a hexamer (adding hexose residues D, E, and F) would not change the conformation of the enzyme, the conformations of the substrate at... 

A half-chair conformation of a crystalline monosaccharide has not been observed. A half-chair conformation for the fourth 2-acetamido-2-deoxy-/3-D-glucopyranosyl residue (residue D) in the lysozyme substrate has not been detected, although, on the basis of model fitting, its presence has been suggested (see p. 96). 2-Acetamido-2-deoxy-/3-D-glucosyl groups were added to a molecular model constructed by use of data obtained from the nature of the enzyme-trisaccharide complex it was implicit that the lifetime of the half-chair conformation would be quite short. 

In order to show that the origin of this difference is not a function of the particular substrate analogue used, similar NMR relaxation studies have been performed with dimethyl sulfoxide (DMSO)1401 since the crystal structure of the enzyme-NADH-DMSO ternary complex is well resolved.1366 From the relaxation data, the distance between the methyl protons of DMSO and Co11 was calculated to be 8.9 0.9 A, again too great for direct coordination of the sulfoxide group to the metal ion. Since the cobalt enzyme appears to be functionally similar to the native enzyme, the difference is unlikely to be a direct result of substitution. One possibility is that there may actually be a difference between the solution and crystalline structure of the enzyme ternary complex, particularly since it is well established that the crystalline enzyme is 1000 times less active than in solution.1402... 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

A° resolution (48). The structures of the enzyme in complex with ATP and ADP-Glc were determined to 2.6 and 2.2 A° resolution, respectively. Ammonium sulfate was used in the crystallization process and was found tightly bound to the crystalline enzyme. It was also shown that the small-subunit homotetrameric potato tuber ADP-Glc PPase was also inhibited by inorganic sulfate with the I0.5 value of 2.8 mM in the presence of 6-mM 3-PGA (48). Sulfate is considered as an analog of phosphate, which is the allosteric inhibitor of plant ADP-Glc PPases. Thus, the atomic resolution structure of the ADP-Glc PPase probably presents a conformation of the allosteric enzyme in its inhibited state. The crystal structure of the potato tuber ADP-Glc PPase (48) allows one to determine the location of the activator and substrate sites in the three-dimensional structure and their relation to the catalytic residue Aspl45. The structure also provides insights into the mechanism of allosteric regulation. 

Considerable knowledge exists about the nature of the active site of enzymes, their secondary, tertiary, and quaternary structures. In the case of those enzymes that have been obtained in crystalline form and subjected to X-ray analysis, conformational appearance has been deduced. However, the geometry of such an active site, as elucidated on an isolated crystalline enzyme, need not necessarily be complementary to the natural substrate, or drug molecule, to complex and interact with it. Since we visualize such a site as being flexible rather than rigid, the substrate or drug can induce such a complementary fit. 

The nature of the Ngose reaction is described with respect to electron donation, energy requirement, and reduction characteristics, with particular analysis of the seven classes of substrates reducible by N20se, a complex of a Mo-Fe and Fe protein. Chemical and physical characteristics of Fe protein and crystalline Mo-Fe protein are summarized. The two-site mechanism of electron activation and substrate complexation is further developed. Reduction may occur at a biological dinuclear site of Mo and Fe in which N2 is reduced to NH3 via enzyme-bound diimide and hydrazine. Unsolved problems of electron donors, ATP function, H2 evolution and electron donation, substrate reduction, N20se characteristics and mechanism, and metal roles are tabulated, Potential utilities of N2 fixation research include in-creased protein production and new chemistry of nitrogen. 

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