3pcd
From Proteopedia
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PROTOCATECHUATE 3,4-DIOXYGENASE Y447H MUTANT
Overview
The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408, (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray, crystallographic studies have shown that Tyr447 is dissociated from the, Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here, by site-directed mutation of this residue to His (Y447H), the first such, mutation reported for an aromatic ring cleavage dioxygenase containing, Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181), is essentially unchanged from that of the native enzyme outside of the, active site region. The side chain position of His447 is stabilized by a, His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of, His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827, appears to be replaced by a CO32-, thereby retaining the overall charge, neutrality and coordination number of the Fe3+ center. Quantitative metal, and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of, the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than, that of the native enzyme. Single-turnover kinetic analysis of the, Y447H-catalyzed reaction reveals that slow substrate binding accounts for, the decreased kcat. Three new kinetically competent intermediates in this, process are revealed. Similarly, the product dissociation from Y447H is, slow and occurs in two resolved steps, including a previously unreported, intermediate. The final E.PCA complex (ES4) and the putative E.product, complex (ESO2*) are found to have optical spectra that are, indistinguishable from those of the analogous intermediates of the, wild-type enzyme cycle, while all of the other observed intermediates have, novel spectra. Once the E.S complex is formed, reaction with O2 is fast., These results suggest that dissociation of Tyr447 occurs during turnover, of 3,4-PCD and is important in the substrate binding and product release, processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex, by either dissociation or mutagenesis, the O2 attack and insertion steps, proceed efficiently, suggesting that Tyr447 does not have a large role in, this phase of the reaction. This study demonstrates a novel role for Tyr, in a biological system and allows evaluation and refinement of the, proposed Fe3+ dioxygenase mechanism.
About this Structure
3PCD is a Protein complex structure of sequences from Pseudomonas putida with CO3, FE and BME as ligands. Active as Protocatechuate 3,4-dioxygenase, with EC number 1.13.11.3 Known structural/functional Sites: , , , , , , , , , , and . Full crystallographic information is available from OCA.
Reference
The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates., Frazee RW, Orville AM, Dolbeare KB, Yu H, Ohlendorf DH, Lipscomb JD, Biochemistry. 1998 Feb 24;37(8):2131-44. PMID:9485360
Page seeded by OCA on Tue Dec 18 20:41:00 2007
