Structural basis for the inactivation of Thermus thermophilus proline dehydrogenase by N-propargylglycine.
The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 A resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the -amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25 degrees , which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this alpha,beta-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely.
White
Tommi A
TA
Department of Chemistry , University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Johnson
William H
WH
Whitman
Christian P
CP
Tanner
John J
JJ
D000480:Alkynes / Q000737:chemistry* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011394:Proline Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D000480:Alkynes; D004791:Enzyme Inhibitors; C009055:propargylglycine; D011394:Proline Oxidase; D005998:Glycine