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J. Biol. Chem., Vol. 279, Issue 1, 341-347, January 2, 2004

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A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase^*

Jongchul Yoon, Bora Oh, Kyunggon Kim, Jungeun Park, Dohyun Han, Kyeong Kyu Kim¶, Sun-Shin Cha||, Dongsoon Lee**, and Youngsoo Kim

From the Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, 28 Yongon-Dong, Seoul 110-799, ^¶Department of Molecular Cell Biology, Center for Molecular Medicine, SBRI, Sungkyunkwan University, School of Medicine, Suwon 440-746, ^||Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, ^**Department of Clinical Pathology, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, and Cancer Research Institute, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799 Korea

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an -subunit, a spacer sequence, and a -subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with - and -subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1 onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1 to generate a strong nucleophile and to direct the OG atom of the Ser-1 to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.

Received for publication, August 21, 2003 , and in revised form, October 7, 2003.

^* This work was supported by Seoul National University Hospital Research Fund Grant 09-2003-011-0. This work was also supported by a research grant from the Cancer Research Institute, Seoul National University (2003). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 82-2-3668-7950; Fax: 82-2-741-7947; E-mail: biolab{at}snu.ac.kr.

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