Phosphorylase crystallization

Shi W, Ting LM, Kicska GA, Lewandowicz A, Tyler PC, Evans GB, Furneaux RH, Kim K, Almo SC, Schramm VL (2004) Plasmodium falciparum purine nucleoside phosphorylase crystal structures, immucillin inhibitors, and dual catalytic function. J. Biol. Chem. 279 18103-18106... 

Meyerhof s discovery in yeast extracts of a phosphorylase (which he named hexokinaseP) led to more detailed investigations of the specificity of this enzyme. The enzyme, which has been crystallized,89 converts D-glucose,... 

Nucleoside phosphorylases that catalyse the reversible cleavage of purine nucleosides to the free bases and ribose-1-phosphate are found in most cells, although a phosphorylase that will cleave adenosine has so far been identified only in bacteria. Recent studies have shown that ribo- and 2 -deoxyribofurano-syltransferase activity is associated with phosphorylase activity [19, 23., 222] and that both activities reside in one enzyme, which can be converted from one form to the other by substrate or product binding [20]. Upon crystallization of the enzyme from human erythrocytes a marked decrease in the ribosyl transfer reaction was observed [21b]. 

Figure 16.4 A plot showing likely crystal stability with increasing particle size based on the observation that for a given surface roughness (e.g. 10 A) the likely contact area is proportional to the particle radius R, whilst the dispersive force is proportional to (i.e. the particle mass). PPb = glycogen phosphorylase b, FMDV = foot-and-mouth disease virus PRDl = PRDl bacteriophage.

S. C. Almo, and V. L. Schramm, Over-the barrier transition state analogues and crystal stmcture with Mycobacterium tuberculosis purine nucleoside phosphorylase, Biochemistry, 42 (2003) 6057-6066. 

An extreme example of the reduction in radiation damage is that of data collection at the SRS on purine nucleoside phosphorylase. On a conventional source usually a crystal can give only one 3 A resolution still photograph before the crystal suffers serious damage. At the synchrotron three crystals will give a comptete set of 4 equivalent reflections to a resolution limit of 3 A (see case study below)... 

Case Study C Large Solvent Content Crystals of Human Erythrocyte Purine Nucleoside Phosphorylase. 

Fig. 3. The structure of purine nucleoside phosphorylase determined using synchrotron radiation to a resolution limit of 3 A (crystals r32, a = 99.2 A = 92.1)...

Fig. 8. A full white beam Laue diffraction pattern recorded from a crystal of glycogen phosphorylase b, and (P4(3)2(l)2 a = b = 128.8 A, c= 116.2 A) using a 0.2 mm diameter collimator the exposure time was 0.5 s...

Fig. 14.1. Ribbon structure (magenta) of the phosphorylase kinase crystal structure 2PHK (20) bound with ATP (green carbons, colored by atom type) and substrate peptide (light blue ribbon). The N- and C-terminal lobes are highlighted the hinge region is shown in cyan, the a-C helix in gray, and the -loop in orange.

Cook WJ, Ealick SE, Bugg CE, Stoeckler JD, Parks RE Jr. Crystallization and preliminary X-ray investigation of human erythrocytic purine nucleoside phosphorylase. JBiol Chem 1981 256 4079-80. 

In the analysis of the structural data of other protein kinases, it is noted that only cAPK has been crystallized with its specific peptide inhibitor. Nevertheless, three other structures of protein kinases compared with the structure of the cAPK-PKI complex provide substantial evidence for the conservation of the substrate binding cleft. The substrate binding cleft of the phosphorylase kinase structure has been analyzed in detail and it is clear that all amino acids of the known specific substrate can be built into the PKI model and all required corresponding charges can be found in the cleft of the phosphorylase kinase structure. In the CK-1 structure determined without a peptide, the requirement of the peptide specificity resides on the P-3 site, which has to be phosphorylated. An analysis of the surface charges of the cleft of the CK-1 structure reveals the exact correspondence of the residues required to interact with a phosphorylated substrate at this site. 

In many cases, the crystal retains enzymatic activity. In some cases, the activity of the enzyme in the crystal is the same as that in solution. The methods used for initiating reactions for study by the Laue method are used to measure activity. For example, pH-jump the acylenzyme indolylacryloyl-chymotrypsin was crystallized at a pH at which it is stable. On changing the pH to increase the reactivity, the intermediate was found to hydrolyze with the same first-order rate constant as occurs in solution the reactions of crystalline ras p21 protein, glycogen phosphorylase, and chymotrypsin have been initiated by photolysis.52 Glyceraldehyde 3-phosphate dehydrogenase has also identical reaction rates in the crystal and solution under some conditions.53... 

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. 

Louise Johnson and her coworkers have determined the crystal structures of T and the R forms of muscle phosphorylase b and the R form of phosphorylase a. In parallel with this work, Robert Fletterick and coworkers determined the structure of the T form of muscle phosphorylase a. The crystal structures provide an incisive look at the structural changes that accompany the transitions from the T to the R conformation and from the nonphosphorylated form of the enzyme to the phosphorylated form. 

A shattering experience. Crystals of phosphorylase a grown in the presence of glucose shatter when a substrate such as glucose 1-phosphate is added. Why ... 

E.D. Lowe, M.E. Noble, V.T. Skamnaki, N.G. Oikonomakos, DJ. Owen, and L.N. Johnson. 1997. The crystal structure of a phosphorylase kinase peptide substrate complex Kinase substrate recognition EMBOJ. 16 6646-6658. (PubMed)... 

L. N. Johnson, K.R. Acharya, M.D. Jordan, and P.J. McLaughlin. 1990. Refined crystal structure of the phosphorylase-heptulose-2-phosphate-oligosaccharide-AMP complex J. Mo/. Biol. 211 645-661. (PubMed)... 

---