Glucose-6 phosphate mutants

Abbreviations Abe, abequose Man, mannose Rha, rhamnose Gal, galactose Gic. glucose GlcN Ac, N-acetyl-glucosamine Hep, / lycero-/)-manno/joctulosonic acid Etn, ethanolamine FA. fatty acids-, Gy, phosphate. Mutants that produce incomplete polysaccharide are designated Ra to Re and are indicated by the solid lines SI. PS refers to wild type I. PS. Position and number of fa tty acids linked to lipid A have been updated by Westpha et al. (46). 

Fourthly, biotransformations have been used for the synthesis of 3-deoxy-2-glyculosonic acids, using whole cells or purified enzymes. For instance, 3-deoxy-D-araZu rao-heptulosonic acid (DAH) and its 7-phosphate (DAHP, 122) have been produced directly from D-glucose by mutants of E. coli JB-5, that lack dehydroquinate synthase, the enzyme that converts DAHP into the cyclic intermediate dehydroquinic acid (DHQ, Scheme 14). Both DAH and DAHP are secreted into the medium. The dephosphorylated product could be generated in vivo by a phosphatase acting on DAHP.312... 

D-Glucose PGI1 mutant is deficient in D-glucose phosphate isomerase 344... 

H8. Hirono, A., Kuhl, W., Gelbart, T., Forman, L., Fairbanks, V. F., and Beutler, E., Identification of the binding domain for NADP of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants. Proc. Natl. Acad. Sci. U.S.A. 86,10015-10017 (1989). 

M8. Marks, P. A., Szeinberg, A., and Banks, J., Erythrocyte glucose-6-phosphate dehydrogenase of normal and mutant human subjects. ]. Biol. Chem. 236, 10-17 (1961). 

The results of site-directed mutagenesis studies are generally consistent with this model.583 Table 4.3 summarizes the kinetic parameters of the mutant enzymes where the five labeled lysyl residues are individually replaced by Gin. The Lys-367/Gin enzyme was almost completely inactive. The Lys-263/ Gin enzyme had slight activity with decrease in affinity to pyrophosphate and glucose 1-phosphate. The Lys-329/Gin enzyme also had considerably lower affinity to the two substrates, but possessed a Kraax value comparable to the wild-type enzyme. The replacements of Lys-409 and Lys-410 by Gin did not change the kinetic properties of the enzyme. The kinetic properties of the Lys-329/Gin enzyme suggest that Lys-329 interacts with pyrophosphate after UDP-glucose binds to the active site. 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

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