Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Research Abstract Details 

Research Abstract Table of Contents

Jump to the:

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Abstract Text:

Chi-Lin Tsai,Kuppan Gokulan,Pablo Sobrado,James C Sacchettini,Paul F Fitzpatrick,

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Publishing Authors By Initials

CL Tsai,K Gokulan,P Sobrado,JC Sacchettini,PF Fitzpatrick,

For similar investigative techniques: genetic techniques: sequence alignment research abstracts see: investigative techniques: genetic techniques: sequence alignment research

PUBMED ID PMID:

MEDLINE DATE:

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 7844-51

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 12

MONTH: 06

YEAR: 2007

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370623

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Keywords Mesh Terms:

KEYWORDS: Sequence Alignment

MESH TERMS: enzymology

Chemical & Substance for Abstract: Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base. Information

Substance Name: L-Lactate Dehydrogenase (Cytochrome)

Registry Number: EC 1.1.2.3

Grant and Affiliation Information for Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

AFFILIATION: Department of Chemistry, Texas A&M University, College Station, Texas 77843-2128, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM58698

ACRONYM: GM

MEDLINETA: Biochemistry

REFSOURCE:

DATABASENAME:

ACCESSION NUMBER:

Number Hits: 0

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base Related Publications

• A grim future foretold.
• Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor.
• Behavioral sensitivity to interaural time differences in the rabbit.
• Bereavement among faculty members in a university setting.
• Connections of functional areas in the mustached bat's auditory cortex with the auditory thalamus.
• Cortical cartography revisited: A frequency perspective on the functional architecture of visual cortex.
• Detection of simultaneous movement at two human arm joints.
• Development of the Oxford ankle foot questionnaire: finding out how children are affected by foot and ankle problems.
• Direct spectroscopic evidence for a high-spin Fe(IV) intermediate in tyrosine hydroxylase.
• Endogenous growth factors as cosmeceuticals.
• Global theme issue on poverty and human development.
• ISI Web of Knowledge:New Author Searching Capability and Reports Features.
• Keeping your cool while changing your job.
• Laser resurfacing today and the "cookbook" approach: a recipe for disaster? A comment.
• Levels of autonomy of nurse practitioners in an acute care setting.
• Luminance-evoked inhibition in primary visual cortex: a transient veto of simultaneous and ongoing response.
• Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.
• Mechanistic studies of the flavoenzyme tryptophan 2-monooxygenase: deuterium and 15N kinetic isotope effects on alanine oxidation by an L-amino acid oxidase.
• Neural and behavioral sensitivity to interaural time differences using amplitude modulated tones with mismatched carrier frequencies.
• Oxidation of 2-Cys-peroxiredoxins by arachidonic acid peroxide metabolites of lipoxygenases and cyclooxygenase-2.
• Physiological and metabolic effects of prophylactic treatment with the osmolytes glycerol and betaine on Bos indicus steers during long duration transportation.
• Posttraumatic stress disorder and posttraumatic stress disorder-like symptoms and mild traumatic brain injury.
• Safety and acceptability of implantation of internal cardioverter-defibrillators under local anesthetic and conscious sedation.
• Socioeconomic status, psychosocial health and health behaviours among Hungarian adolescents.
• The end of the road for the campaign against MMR.
• The gender conundrum.
• The reciprocal relationship between melanization and tyrosinase activity in melanosomes (melanin granules).
• Two-Year Treatment With Denosumab (AMG 162) in a Randomized Phase 2 Study of Postmenopausal Women With Low BMD.
• Vision and cortical map development.
• Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC.