Anatomy of a Simple Acyl Intermediate in Enzyme Catalysis: Combined Biophysical and Modeling Studies on Ornithine Acetyl Transferase

Aman Iqbal, Ian J. Clifton, Maria Bagonis, Nadia J. Kershaw, Carmen Domene Nunez, Timothy D.W. Claridge, Christopher W. Wharton, Christopher J. Schofield

Research output: Contribution to journalArticlepeer-review

11 Citations (SciVal)


Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the α-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; 13C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-α-acetyl-l-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm−1. Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm−1 IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm−1 IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.
Original languageEnglish
Pages (from-to)749-757
Number of pages9
JournalJournal of the American Chemical Society
Issue number2
Publication statusPublished - 1 Jan 2009




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