The role of histidine residues in the HXGH site of CTP:phosphocholine cytidylyltransferase in CTP binding and catalysis.

The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10743-10750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP-binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100-fold lower than that of wild-type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition-state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the tau nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition-state stabilization.

Substitution of serine for glycine-91 in the HXGH motif of CTP:phosphocholine cytidylyltransferase implicates this motif in CTP binding.

The effect of mutations in the proposed catalytic domain of CTP:phosphocholine cytidylyltransferase was investigated by constructing the single mutants CT-S91 and CT-C114 from the double mutant CT-S91C114, previously shown to have 4-fold lower than wild-type activity [Walkey, C.R., Kalmar, G. B., & Cornell, R. B. (1994) J. Biol. Chem. 269, 5742-5749]. The constructs were overexpressed in COS cells. The mutation Gly-91 to Ser-91 was found to be responsible for the decreased activity, whereas Ser-114 to Cys-114 had no effect. An alanine substitution at position 91, CT-A91, had a lesser effect on cytidylyltransferase activity. CT-S91 and CT-WT were purified from COS cells, and their kinetic constants were determined. CT-S91 had a 4-fold lower Vmax, and a K(m) for CTP 25-fold higher than the wild-type enzyme, suggesting that substitution of Gly-91 with serine interferes with CTP binding. The K(m) for phosphocholine was not affected in the CT-S91 mutant. There was no difference in the chymotrypsin sensitivities of CT-S91 and CT-WT, indicating that the mutation did not cause a global change in protein structure. However, the CT-S91 activity was more susceptible to inhibition by the denaturant urea than that of CT-WT, indicative of a perturbation of the active site folding. Gly-91 resides in the local sequence HSGH, which has been proposed to be a CTP-binding motif in the novel cytidylyltransferase superfamily [Bork, P., Holm, L., Koonin, E.V., & Sander, C. (1995) Proteins: Struct., Funct., Genet. 22, 259-266]. Our results represent the first experimental validation of this hypothesis.

Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of phenyl-Sepharose for the affinity purification of proteinases.

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.