Crystallization, enzymes

Once mounted in the diffractometer, the crystal is irradiated with X rays, usually so-called Cu/Cg radiation with a wavelength of 0.154 nm. When the X rays strike the enzyme crystal, they interact with electrons in the molecule and are scattered into a diffraction pattern, which, when delected and visualized, appears as a scries of intense spots against a null background. 

Williams and coworkers have reported a DKR of ot-bromo [56a] and a-chloro esters [56b]. In the latter case, the KR is catalyzed by commerdally available cross-linked enzyme crystals derived from Candida cylindracea lipase. The racemization takes place through halide 5 2 displacement. The DKR is possible because the racemization of the substrate is faster than that of the produd (carboxylate). For the ester, the empty ii (C=0) orbital is able to stabilize the Sn2 transition state by accepting... 

The most effective of these include immobilization [80], lipid coating [81], surfactant coating [82], use of cross-linked enzyme crystals [83], cross-linked enzyme aggregates [84], and membrane reactors [85]. 

Enzyme crystallization, with or without cross-linking... 

Metalloenzymes, 33 40, see also Enzymes crystal structure, 44 230-258 DNA repair, 45 251 inhibitors, 36 40-42 molybdenum, 45 2, 53, 60-63 nuleic acid hydrolysis, 45 251-252 superoxide dismustases and, 45 130 zinc-containing, hard and soft acid-base behavior, 42 103-109 Metalloids... 

Crosslinking of enzyme crystals with e.g. glutaraldehyde results in the so-called CLECs produced by Altus Biologies inc. 

Figure 9.2 A schematic presentation of different types of enzyme preparations used in non conventional media, a enzyme powder, b enzyme crystals c enzyme on a porous support d covalently modified enzyme dissolved in the solvent e enzyme solubilised by surfactant ...

Crystals constitute the most concentrated form of enzyme and they can therefore be attractive as catalysts. In the crystallisation of a crude enzyme preparation a considerable purification of the enzyme can be achieved which is a further advantage. In order to stabilise the enzyme crystals to make them useful catalysts they are often crosslinked with bifunctional reagents such as glutaraldehyde. Very high catalytic activity and stability has been reported for these crosslinked enzyme crystals, some of which are commercially available (Margolin, 1996). 

For CPA the geometries of both a cylinder and slab were analyzed. At given experimental conditions, Carman-Haul equations predict substantially different curves for the assumption of slab and cylinder geometries. The fact that both equations gave values of D0, EA, AS, and AG in close agreement (Table II) showed that the values obtained for the dimensions of the enzyme crystals (Table I) were reasonable. Of the two geometries, the cylinder is probably the more reliable since the diffu-... 

But where there is an equilibrium among two or more conformations of the enzyme in solution, crystallization may select out only one of the conformations. a-Chymotrypsin has a substantial fraction of an inactive conformation present under the conditions of crystallization, but only the active form of the enzyme crystallizes. An allosteric effector molecule that changes the conformation of the protein in solution may have no effect on the crystalline protein, as, for example, with phosphorylase b.5A The enzyme is frozen in one conformation, with the crystal lattice forces preventing any conformational change. On the other hand, the addition of an effector to phosphorylase a causes the crystals first to crack and then to anneal, giving crystals of the enzyme in a second conformation. 

Some of the physicochemical parameters of the enzyme, as determined on the Kunitz preparation (S), are summarized in Table I. Sedimentation equilibrium analysis of molecular weight carried out on the enzyme crystallized from ammonium sulfate gave a value of 71,000 (5), in fair agreement with the value of 63,000 obtained by sedimentation velocity measurements on the Kunitz preparation (10). The enzyme has been reported to dissociate into subunits in the presence of sodium dodecyl... 

Analysis in two different laboratories of the amino acid composition of the enzyme crystallized from ethanol gave similar results with the exception of the values for cysteine. Hausmann (11) found only a trace, but Eifler et al. (12) reported 3 cysteine residues per mole of 60,000 molecular weight. The latter investigators found that three different sulfhydryl reagents reacted with the enzyme in amounts approaching one mole per mole of enzyme. A total of three -SH per mole were found after reduction with sodium borohydride. The investigators suggested that the molecule contains one -S-S- and one -SH, which, if true, forbids identity of subunits. 

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