Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.
The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.
Haouz
Ahmed
A
Unité de Biochimie Structurale, URA 2185 du CNRS, Biologie Structurale et Agents Infectieux, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
Vanheusden
Veerle
V
Munier-Lehmann
Hélène
H
Froeyen
Mattheus
M
Herdewijn
Piet
P
Van Calenbergh
Serge
S
Delarue
Marc
M
D020134:Catalytic Domain; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase