Abstract
Histidine 30 in human manganese superoxide dismutase (MnSOD) is located at a site partially exposed to solvent with its side chain participating in a hydrogen-bonded network that includes the active-site residues Tyr(166) and Tyr(34) and extends to the manganese-bound solvent molecule. We have replaced His(30) with a series of amino acids and Tyr(166) with Phe in human MnSOD. The crystal structure of the mutant of MnSOD containing Asn(30) superimposed closely with the wild type, but the side chain of Asn(30) did not participate in the hydrogen-bonded network in the active site. The catalytic activity of a number of mutants with replacements at position 30 and for the mutant containing Phe(166) showed a 10-40-fold decrease in k(cat). This is the same magnitude of decrease in k(cat) obtained with the replacement of Tyr(34) by Phe, suggesting that interrupting the hydrogen-bonded active-site network at any of the sites of these three participants (His(30), Tyr(34), and Tyr(166)) leads to an equivalent decrease in k(cat) and probably less efficient proton transfer to product peroxide. The specific geometry of His(30) on the hydrogen bond network is essential for stability since the disparate mutations H30S, H30A, and H30Q reduce T(m) by similar amounts (10-16 degrees C) compared with wild type.
Highlights
IntroductionThe maximal velocity of catalysis has rate-contributing proton transfer steps as determined by the solvent hydrogen isotope effect of 2.1 for the turnover number kcat for catalysis by T. thermophilus manganese superoxide dismutase (MnSOD) [3], and the enhancement of maximal velocity by proton donors in solution observed for catalysis by the very similar Escherichia coli iron superoxide dismutase [4]
Structure of H30N manganese superoxide dismutase (MnSOD)—Unlike the native and other functional mutant structures of MnSOD, which crystallize in space group P21212 and have two MnSOD subunits in the asymmetric unit, H30N crystallized in space group P212121 and had four MnSOD subunits in the asymmetric unit
A hydrogen-bonded array involving residues 30 and 166 that may be involved in the protonation of product peroxide is not as extensive in H30N and Y166F as it is in wild type
Summary
The maximal velocity of catalysis has rate-contributing proton transfer steps as determined by the solvent hydrogen isotope effect of 2.1 for the turnover number kcat for catalysis by T. thermophilus MnSOD [3], and the enhancement of maximal velocity by proton donors in solution observed for catalysis by the very similar Escherichia coli iron superoxide dismutase [4]. This protonation promotes the dissociation of product from the enzyme and perhaps decreases the extent of product inhibition. The side chain of Tyr forms a hydrogen bond with metal-bound azide, a substrate analog, in MnSOD from T. thermophilus [9]
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