Studies of the Shikimate Pathway Enzyme, 3-Dehydroquinase

Deka, Ranjit Kumar (1993) Studies of the Shikimate Pathway Enzyme, 3-Dehydroquinase. PhD thesis, University of Glasgow.

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1. Chemical modification with diethylpyrocarbonate (DEPC) and kinetic analyses have established a role for a single active site histidine residue as the general base involved in the mechanism of the type I dehydroquinase of E. coli. 2. Differential peptide mapping of DEPC-modified dehydroquinase in the absence and presence of substrate identified a peptide containing the active site histidine residue. 3. Sequence comparison of the active site peptide with other type I dehydroquinases allowed the identification of the active site histidine as His-143 in the E. coli. enzyme sequence. 4. The type II dehydroquinase of A. nidulans shows very different properties (eg. Km,Vmax, Vmax versus pH plots) from those found for the type I enzyme from E. coli. 5. The type II enzyme from A. nidulans does not work through a Schiff base adduct since it cannot be inhibited by treatment with substrate plus sodium borohydride. 6. The type II dehydroquinase from A. nidulans like the type I enzyme from E. coli, has at least one essential active site histidine residue. 7. The type II dehydroquinase is very susceptible to phenylglyoxal inactivation indicating the involvement of an arginine residue(s) in its mode of action. 8. The previously published purification of the P. sativum dehydroquinase-shikimate dehydrogenase has been scaled up and the yield improved. The purified P. sativum dehydroquinase has a similar Km and pH optimum to the type I dehydroquinase from E. coli. 9. A borohydride trapping experiment established that the dehydroquinase from P. sativum is a Schiff base forming type I enzyme. Chemical modification with the histidine specific reagent DEPC suggests that, like the E. coli type I enzyme, there is a histidine residue essential for catalytic activity. 10. The shikimate dehydrogenase activity of the P. sativum bifunctional dehydroquinase-shikimate dehydrogenase was not inhibited by the dehydroquinate/sodium borohydride treatment which completely inactivated the dehydroquinase activity. This establishes that the shikimate dehydrogenase and dehydroquinase active sites are spatially distinct. 11. N-terminal equencing of the intact P. sativum bifunctional dehydroquinase-shikimate dehydrogenase and of peptides generated by Cleveland mapping provided 157 amino acid residues of sequence. All of the sequences determined showed similarities to regions of either the dehydroquinase or the shikimate dehydrogenase of E. coli. 12. The N-terminal sequence of the intact P. sativum bifunctional dehydroquinase-shikimate dehydrogenase is clearly homologous to the N-terminal region of E. coli. dehydroquinase; this clearly establishes that in the bifunctional polypeptide the dehydroquinase domain is N-terminal. 13. Using the peptide sequences to aid primer design a simple PCR strategy was developed to amplify fragments of the cDNA encoding the entire dehydroquinase coding region and part of the shikimate dehydrogenase coding region of the P. sativum bifunctional dehydroquinase-shikimate dehydrogenase. 14. The complete amino acid sequence of the dehydroquinase domain of the P. sativum bifunctional dehydroquinase-shikimate dehydrogenase was deduced from the nucleotide sequence of the DNA fragments. At the amino acid level there was from 23 to 28% identity between the P. sativum dehydroquinase domain and the other known type I dehydroquinase domains; both the known active sites residues, His-143 and Lys-170 (numbering is according to the E. coli enzyme), are conserved in the P. sativum enzyme. Another residue, Asp-144, which is conserved in all previously studied type I dehydroquinases, is not conserved in the P. sativum enzyme which implies that this residue is not involved in the mechanism of action.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J R Coggins
Keywords: Biochemistry
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-76370
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:20
Last Modified: 19 Nov 2019 15:20

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