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Title: NMR characterisation of ligand-bound dimethylarginine dimethylaminohydrolase
Author: Rasheed, M.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2012
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This thesis describes an extensive spectroscopic investigation of the dynamics and ligand binding properties of the enzyme dimethylaminoarginine dimethylaminohydrolase from Pseudomonas aeruginosa (PaDDAH). The presumed function of the enzyme is the hydrolysis of asymmetrically side chain‐methylated arginine residues to the products Lcitrulline and (di) methylamine. The homologous mammalian DDAH enzymes counteract the inhibition of nitric oxide synthase (NOS) by asymmetrically methylated arginines (e.g. asymmetric dimethylarginine, ADMA). The precise functional role of prokaryotic DDAH remains obscure. The 3D structure of DDAH is comprised of a single polypeptide chain of 254 amino acid residues folded into a relatively unusual five‐bladed α/β‐propeller topology. The active site of the enzyme is located at the core of the fold, which substrates must access via a channel capped by a loop ‘lid’ structure. This thesis describes the application of nuclear magnetic resonance (NMR) spectroscopy and other methods to characterise the substrate‐ and product‐bound states of variant forms of PaDDAH. Using an engineered non‐associating form of the enzyme (R40E, R98H PaDDAH, referred to as DM‐PaDDAH) as a platform, the citrulline‐saturated states of DM‐PaDDAH and a putative ‘hydrolase‐dead’ form (R40E, R98H, C249S PADDAH; TM‐PaDDAH) have been obtained. The TM‐PaDDAH exhibited low but finite activity towards standard substrates, prohibiting a full characterisation of the substrate‐bound enzyme. Multiple‐field nitrogen‐15 nuclear relaxation analysis of the apo‐ and citrulline‐bound states of DM‐PaDDAH suggest that ligand binding is accompanied by a disorder‐order transition for a section of the active site ‘lid’ between residues 17‐21. However mutation of the ‘gatekeeper’ lid residue Leu18, which ‘sits down’ on the bound ligand in the crystal structure of citrulline‐bound PaDDAH, to alanine has relatively little influence on the kinetic properties of the enzyme suggesting that lid‐opening and product release are not the rate‐limiting steps in the hydroylase mechanism. Finally the prospect for developing a citrulline/ADMA amino acid sensor based upon the attachment of an environmentally sensitive fluorophore to a Cys side chain substituted at the native PaDDAH Ser20 position was explored by stopped‐flow and other methods.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available