1qnm

From Proteopedia

Revision as of 16:48, 12 November 2007

HUMAN MANGANESE SUPEROXIDE DISMUTASE MUTANT Q143N

Overview

Structural and biochemical characterization of the nonliganding residue, glutamine 143 near the manganese of human Mn superoxide dismutase, (hMnSOD), a homotetramer of 22 kDa, reveals a functional role for this, residue. In the wild-type protein, the side-chain amide group of Gln 143, is about 5 A from the metal and is hydrogen-bonded to Tyr 34, which is a, second prominent side chain adjacent to the metal. We have prepared the, site-specific mutant of hMnSOD with the conservative replacement of Gln, 143 --> Asn (Q143N). The crystal structure of Q143N shows that the, side-chain amide nitrogen of residue 143 is 1.7 A more distant from the, manganese than in the wild-type enzyme. The Tyr 34 side-chain hydroxyl in, Q143N is also moved to become 0.6 A more distant from the metal due to an, additional water molecule. Differential scanning calorimetry showed that, Q143N is slightly more stable than the wild-type enzyme with Tm for the, main unfolding transition increased by 2 degrees C to 90.7 degrees C., Pulse radiolysis and stopped-flow spectrophotometry reveal that unlike, wild-type hMnSOD, which is strongly inhibited by peroxide, Q143N MnSOD, exhibits no product inhibition even at concentrations of O2. - in the, millimolar range, and its catalysis follows Michaelis kinetics with no, evidence of cooperativity. However, the overall catalytic activity of this, mutant was decreased 2-3 orders of magnitude compared with the wild-type, MnSOD, which can account for its lack of product inhibition. Q143N MnSOD, lacked the visible absorption spectrum typical of wild-type Mn(III)SOD., Also, unlike the wild-type Mn(III)SOD, which is electron paramagnetic, resonance (EPR) silent, Q143N MnSOD has a complex EPR spectrum with many, resonances in the region below 2250 G. We conclude that the Gln 143 -->, Asn mutation has increased the reduction potential of manganese to, stabilize Mn(II), indicating that Gln 143 has a substantial role in, maintaining a reduction potential favorable for the oxidation and, reduction cycles in the catalytic disproportionation of superoxide. A, solvent hydrogen isotope effect near 2 for kcat in catalysis by Q143N, hMnSOD indicates rate-contributing proton transfers to form product, hydroperoxide anion or hydrogen peroxide. The data demonstrate a prominent, role for Gln 143 in maintaining the microenvironment of the manganese and, in efficient catalysis of superoxide dismutation to oxygen and hydrogen, peroxide.

About this Structure

1QNM is a Single protein structure of sequence from Homo sapiens with MN as ligand. Active as Superoxide dismutase, with EC number 1.15.1.1 Structure known Active Site: MNA. Full crystallographic information is available from OCA.

Reference

Probing the active site of human manganese superoxide dismutase: the role of glutamine 143., Hsieh Y, Guan Y, Tu C, Bratt PJ, Angerhofer A, Lepock JR, Hickey MJ, Tainer JA, Nick HS, Silverman DN, Biochemistry. 1998 Apr 7;37(14):4731-9. PMID:9537988

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