Crystalline enzyme-substrate

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-... 

All these results are encouraging for investigators planning to use X-ray diffraction in mixed solvents at subzero temperatures and the rest of the present article will be devoted to a discussion of methods and preliminary results in this field. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, will be described. Limitations and perspectives of the procedure will be discussed critically. First attempts to determine the structure of productive enzyme-substrate intermediates through stop-action pictures will be described, as well as investigations showing that X-ray diffraction at selected normal and subzero temperatures can reveal protein structural dynamics. 

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. 

The turnover reaction of hydrolysis of 2, 3 -CMP could be made negligibly slow at temperatures below -60°C at pH 3-6 in 70% methanol, and below -35°C at pH 2.1. The rate of the catalytic reaction using crystalline enzyme was found to be 50-fold slower than that of dissolved enzyme for cyclic phosphate hydrolysis, and 200-fold slower for dinucleotide hydrolysis (presumably the greater reduction for the larger substrate reflects increased diffusional hindrance by the small solvent chan-... 

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. 

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... 

The development of synchrotron radiation as an X-ray source404 416 418 has permitted accumulation of data for electron density difference maps in less than 1 s and it is expected that such data can eventually be acquired in 1 ps.419-421 If a suitable photochemical reaction can be initiated by a picosecond laser flash, a substrate within a crystalline enzyme can be watched as it goes through its catalytic cycle. An example is the release of inorganic phosphate ions from a "caged phosphate" (Eq. 3-49) and study of the reaction of the released phosphate with glycogen phosphorylase (Chapter 12).422/423... 

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... 

Relative rates of hydrolysis were determined with 0.5 ml reaction mixtures in 0.1 M sodium acetate buffer, pH 5.0, at 37°. Liberated phosphate was measured by the method of C. H. Fiske and Y. SubbaRow [JBC 66, 375 (1925)]. The amounts of enzyme used were 0.22 unit of crystalline enzyme and 0.24 unit of peak II enzyme. The concentration of substrate and inhibitor was 1.0 mM. For inhibitor study, 1.0 mM p-nitrophenyl phosphate was used as substrate. Inhibition was calculated from the amount of p-nitro-phenol released and expressed as fractional inhibition. 

Although many of these general features are correct, these mechanistic conclusions are based upon the assumption that the properties of the enzyme in solution are conserved on crystallization. It appears from Raman studies on arsanilazotyrosine-248 carboxypeptidase that the enzyme exists in solution in a number of different forms.509 It is not certain that the form which crystallizes out is the kinetically active species. Indeed there is evidence that the kinetics of carboxypeptidase in solution differ from those of the enzyme crystals, with the crystalline enzyme being 1000-fold less active with some substrates.510 The interaction of Gly-L-tyr with Mn carboxypeptidase A, studied by 1H NMR techniques, does not involve coordination of the carboxyl group of the substrate, and may well represent a different conformation from the one studied by crystallographic techniques.511 Several conformational forms of the Cd11 carboxypeptidase A have been found in solution, while the enzyme exists in a different form in the crystal state.312... 

Probably the most convincing evidence that crystalline structures can safely be used to draw conclusions about molecular function is the observation that many macromolecules are still functional in the crystalline state. For example, substrates added to suspensions of crystalline enzymes are converted to product, albeit at reduced rates, suggesting that the enzyme s catalytic and binding sites are intact. The lower rates of catalysis can be accounted for by the reduced accessibility of active sites within the crystal, in comparison to solution. 

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 ... 

Formation of Secondary Products and Lipohydroperoxide Destruction. As early as 1945 Holman and Burr (132) found that crude soybean lipoxygenase acting on a number of substrates produced carbonyl-containing material in addition to diene. Holman, as noted above (107), used his crystalline enzyme and found that it was difficult to establish a correspondence between O2 consumption and diene conjugation. The diene concentration always tended to be too low. Privett et al. 123) found that the reaction products varied with enzyme concentration and method of addition. Vioque and Holman 133) identified 9-keto-ll,13- and 13-keto-9,ll-octadecadienoate with the usual hydroperoxides in a reaction carried out with linoleic acid and a relatively large amount of crude soybean lipoxygenase at pH 9. 

Q-Enzyme is the enzyme thought to be responsible for effecting a D-glucosyl exchange reaction in which an a-D-(l- 4)-linkage in amy-lose is converted into an a-D-(l—>6)-linkage. Probably present in all plants, the enzyme was first isolated, and later crystallized, from the potato. Purification was achieved by fractional precipitation with ethanol at low temperatures. Few properties of the crystalline enzyme were detailed, but it was found to be very heat-labile, to be stabilized to some extent by the presence of substrate, and to be activated by inorganic ions. Later experiments have shown this preparation of enzyme to be impure. "... 

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. 

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