Studies on the active-site of Escherichia coli shikimate dehydrogenase

Chackrewarthy, Sureka (1995) Studies on the active-site of Escherichia coli shikimate dehydrogenase. PhD thesis, University of Glasgow.

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Shikimate dehydrogenase (SKDH) catalyses the fourth step of the biosynthetic shikimate pathway, the reversible reduction of 3-dehydroshikimate to shikimate. This Thesis describes the work leading to the identification of three active-site residues in E. coli SKDH. Group specific chemical modification with trinitrobenzene sulfonic acid (TNBS) and kinetic analyses indicated the presence of an essential lysine residue at the active-site. Electrospray mass spectrometry (ESMS) revealed that three lysine residues were modified by TNBS, and in the presence of substrate and coenzyme two were protected from modification. HPLC mass spectrometry (LCMS) identified the three modified residues as Lys-15, Lys- 65 and Lys-217/219. In the presence of substrate and coenzyme Lys-65 was completely protected and Lys-15 was partially protected from modification. Sequence comparison with other known SKDH sequences allowed the identification of Lys-65 as the essential lysine residue. Experiments with methyl shikimate have provided evidence for a role for Lys-65 in substrate binding. Arg-154 was identified as a component of the coenzyme (NADP+) binding site by group specific chemical modification with phenylglyoxal (PGO). Characterisation of PGO modified SKDH by ESMS showed that a PGO modified arginine residue could have either a 1:1 or 2:1 stoichiometry. Protection experiments suggest that Arg-154 interacts with the 2' phosphate group of the adenosine moiety of the coenzyme. Chemical modification with the group specific reagent diethylpyrocarbonate (DEPC) and kinetic analyses indicated the presence of an active-site histidine. Characterisation of DEPC modified SKDH by ESMS showed that in the presence of substrate and coenzyme two histidine residues were protected from DEPC modification. Differential peptide mapping using reverse phase HPLC identified the two protected residues as His-13 and His-253. Sequence comparison with other SKDH sequences identified His-13 as the essential histidine. PH-dependence studies indicated a role for His-13 as a general acid/base in the catalytic reaction of SKDH. As a preliminary step towards solving the three dimensional structure of SKDH small crystals have been obtained.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by funding from the Government of Sri Lankaand the ORS award scheme.
Keywords: Microbiology.
Colleges/Schools: College of Science and Engineering
Supervisor's Name: Coggins, Professor J.R.
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71701
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 16 Jul 2021 12:34
Thesis DOI: 10.5525/gla.thesis.71701

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