3LEO

Structure of human Leukotriene C4 synthase mutant R31Q in complex with glutathione

Summary for 3LEO

• Entry DOI: 10.2210/pdb3leo/pdb
• Related: 2UUH
• Descriptor: Leukotriene C4 synthase, PALMITOLEIC ACID, PALMITIC ACID, ... (9 entities in total)
• Functional Keywords: leukotriene c4 synthase, endoplasmic reticulum, leukotriene biosynthesis, lyase, membrane, nucleus, transmembrane
• Biological source: Homo sapiens
• Cellular location: Nucleus outer membrane; Multi-pass membrane protein: Q16873
• Total number of polymer chains: 1
• Total formula weight: 20518.19

Authors

Niegowski, D.,Martinez-Molina, D.,Rinaldo-Matthis, A.,Nordlund, P.,Haeggstrom, J. (deposition date: 2010-01-15, release date: 2010-10-27, Last modification date: 2023-09-06)

Primary citation

Rinaldo-Matthis, A.,Wetterholm, A.,Martinez Molina, D.,Holm, J.,Niegowski, D.,Ohlson, E.,Nordlund, P.,Morgenstern, R.,Haeggstrom, J.Z.
Arginine 104 is a key catalytic residue in leukotriene C4 synthase.
J.Biol.Chem., 285:40771-40776, 2010

PubMed Abstract: Human leukotriene C(4) synthase (hLTC(4)S) is an integral membrane enzyme that conjugates leukotriene (LT) A(4) with glutathione to form LTC(4), a precursor to the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC(4)S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K(m) for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ(max) at 239 nm, could be detected using R104A or R104S hLTC(4)S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK(a) to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA(4). On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC(4)S by 88 and 70%, respectively, without significantly affecting the k(cat)/K(m) values for GSH, and a crystal structure of R31Q hLTC(4)S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC(4)S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.

PubMed: 20980252
DOI: 10.1074/jbc.M110.105940

Experimental method

X-RAY DIFFRACTION (2.1 Å)