Interrupting the hydrogen bond network at the active site of human manganese superoxide dismutase.
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.
Ramilo
C A
CA
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610, USA.
Leveque
V
V
Guan
Y
Y
Lepock
J R
JR
Tainer
J A
JA
Nick
H S
HS
Silverman
D N
DN
D019943:Amino Acid Substitution; D001216:Asparagine; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006639:Histidine; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016018:Least-Squares Analysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine
D007527:Isoenzymes; D011994:Recombinant Proteins; D014443:Tyrosine; D006639:Histidine; D001216:Asparagine; D013482:Superoxide Dismutase