Crystal Structure of Y34F Mutant Human Mitochondrial Manganese Superoxide Dismutase and the Functional Role of Tyrosine 34,

• DOI: 10.1021/bi972394l
• Publication Date: 2002-07-26T05:17:50Z
• Authors: Yue Guan; Michael J. Hickey; Gloria E. O. Borg...; Robert A. Hallewell; James R. Lepock; Don O'Connor; Yunsheng Hsieh; Harry S. Nick; David N. Silverman; John A. Tainer
• Keywords: Orthorhombic crystal system; Wild type

Tyrosine 34 is a prominent and conserved residue in the active site of manganese superoxide dismutases organisms from bacteria to man. We have prepared mutant containing replacement Tyr → Phe (Y34F) human dismutase (hMnSOD) crystallized it two different crystal forms, orthorhombic hexagonal. Crystal structures hMnSOD Y34F been solved 1.9 Å resolution hexagonal form, denoted as Y34Fhex, 2.2 an Y34Fortho. Both forms give that are closely superimposable with wild-type hMnSOD, phenyl rings wild type very similar orientation. Therefore, Y34F, hydrogen-bonded relay links metal-bound hydroxyl ordered solvent (Mn−OH Gln 143 H2O His 30) broken. Surprisingly, loss hydrogen bonds resulted large increases stability (measured by Tm), suggesting does not play role stabilizing active-site architecture. The functional side chain can be evaluated comparison hMnSOD. had kcat/Km near 109 M-1 s-1, close diffusion-controlled; however, showed kcat for maximal catalysis smaller 10-fold than type. In addition, was more susceptible product inhibition peroxide enzyme. This activity profile breaking hydrogen-bonding at caused suggest proton donor O2• - reduction H2O2 or involved indirectly orienting other residues transfer. Up 100 mM buffers solution failed enhance either protonation cannot release inhibiting bound ion, likely reflecting enclosure shown X-ray structures. increased thermostability equal diffusion-controlled enzymes normal levels evolutionary conservation metalloenzymes reflects constraints extreme rather average cellular conditions. new hypothesis substrate concentrations powerful constraint on may apply most strongly enzyme defenses where ability meet conditions directly affects cell survival.