Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis.

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Research Abstract Details 

Research Abstract Table of Contents

Jump to the:

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Abstract Text:

John B Carrigan,Paul C Engel,John B Carrigan,Paul C Engel,

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-specific forms usually have aspartate at this position. However, replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast, replacement with a more positively charged lysine or arginine, as found in NADPH-dependent members of other dehydrogenase families, allows a more than 1000-fold shift toward NADPH, resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan, W244, was replaced by a smaller alanine (approximately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indicate that improved affinity for the extra phosphate of NADPH is the predominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases.

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Publishing Authors By Initials

JB Carrigan,PC Engel,JB Carrigan,PC Engel,

For similar abstracts research abstracts see: abstracts research

PUBMED ID PMID:

MEDLINE DATE:

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Journal Published:

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

Journal: The FEBS journal

VOLUME: 274

Page Numbers: 5167-74

Journal Abbreviation: FEBS J.

ISSN: 1742-464X

DAY: 10

MONTH: 09

YEAR: 2007

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 101229646

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Keywords Mesh Terms:

KEYWORDS:

MESH TERMS:

Chemical & Substance for Abstract: Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis. Information

Substance Name:

Registry Number:

Grant and Affiliation Information for Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis.

AFFILIATION: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland.

Country: England

AGENCY:

GRANT:

ACRONYM:

MEDLINETA: FEBS J

REFSOURCE:

DATABASENAME:

ACCESSION NUMBER:

Number Hits: 0

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis Related Publications

• A Gata2 intronic enhancer confers its pan-endothelia-specific regulation.
• Administrative issues in child psychiatry.
• Atomic force microscopy and spectroscopy of native membrane proteins.
• Bioinformatic analysis of adhesion proteins.
• Challenges and successes of infusing environmental health content in a nursing program.
• Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate?
• Characterization of mouse CD229 (Ly9), a leukocyte cell surface molecule of the CD150 (SLAM) family.
• Daily and momentary mood and stress are associated with binge eating and vomiting in bulimia nervosa patients in the natural environment.
• Developmental retardation due to urologic disease.
• Duration of the cue-to-pain delay increases pain intensity: a combined EEG and MEG study.
• Effects of phenylalkylamines and benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse inner hair cells.
• Imaging features of posterior mediastinal chordoma in a child.
• Inclusion-body myositis, a multifactorial muscle disease associated with aging: current concepts of pathogenesis.
• Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique.
• Mice deficient for RNA-binding protein brunol1 show reduction of spermatogenesis but are fertile.
• Mutation in the gene encoding lysosomal acid phosphatase (Acp2) causes cerebellum and skin malformation in mouse.
• On the limitations of adiabatic population transfer between molecular electronic states induced by intense femtosecond laser pulses.
• On the road: progress in finding the unique pathway of invariant NKT cell differentiation.
• Pathogenic point mutations in a transmembrane domain of the epsilon subunit increase the Ca2+ permeability of the human endplate ACh receptor.
• Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis.
• Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane.
• Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps.
• Rates of strabismus surgery in the United States: implications for manpower needs in pediatric ophthalmology.
• Role of the subunit composition of central nicotinic acetylcholine receptors for the stimulatory and dopamine-enhancing effects of ethanol.
• Testing the validity of eating disorder diagnoses.
• The influence of tin compounds on the dynamic properties of liposome membranes: a study using the ESR method.
• The orthodontist and the pediatrician.
• Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein.
• Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers.
• Voltage depth profiles of high-frequency oscillations after kainic acid-induced status epilepticus.