Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form

Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: Insertion of valine Cγ into the substrate access channel

Academic Article
uri icon
  • Overview
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

related to degree

  • Stroupe, Margaret, Ph.D. in Biology, Scripps Research 1997 - 2002

authors

  • Hearn, A. S.
  • Stroupe, Margaret
  • Cabelli, D. E.
  • Ramilo, C. A.
  • Luba, J. P.
  • Tainer, John
  • Nick, H. S.
  • Silverman, D. N.

publication date

  • March 2003

journal

  • Biochemistry  Journal

abstract

  • Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.

subject areas

  • Amino Acid Substitution
  • Azides
  • Binding Sites
  • Carbon
  • Catalysis
  • Catalytic Domain
  • Crystallization
  • Crystallography, X-Ray
  • Histidine
  • Humans
  • Manganese
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Substrate Specificity
  • Superoxide Dismutase
  • Thermus thermophilus
  • Valine
scroll to property group menus

Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi0266481

PubMed ID

  • 12627943
scroll to property group menus

Additional Document Info

start page

  • 2781

end page

  • 2789

volume

  • 42

issue

  • 10

©2021 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support