PMID- 9878450
OWN - NLM
STAT- MEDLINE
DCOM- 19990311
LR  - 20131121
IS  - 0022-2836 (Print)
IS  - 0022-2836 (Linking)
VI  - 285
IP  - 2
DP  - 1999 Jan 15
TI  - Insights into the mechanism of domain closure and substrate specificity of 
      glutamate dehydrogenase from Clostridium symbiosum.
PG  - 875-85
AB  - Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine 
      dehydrogenase (LeuDH) have suggested that two substitutions, deep within the 
      amino acid binding pockets of these homologous enzymes, from hydrophilic residues 
      to hydrophobic ones are critical components of their differential substrate 
      specificity. When one of these residues, K89, which hydrogen-bonds to the 
      gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by 
      site-directed mutagenesis to a leucine residue, the mutant enzyme showed 
      increased substrate activity for methionine and norleucine but negligible 
      activity with either glutamate or leucine. In order to understand the molecular 
      basis of this shift in specificity we have determined the crystal structure of 
      the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure 
      suggests that further subtle differences in the binding pocket prevent the mutant 
      from using a branched hydrophobic substrate but permit the straight-chain amino 
      acids to be used as substrates. The three-dimensional crystal structure of the 
      GluDH from C. symbiosum has been previously determined in two distinct forms in 
      the presence and absence of its substrate glutamate. A comparison of these two 
      structures has revealed that the enzyme can adopt different conformations by 
      flexing about the cleft between its two domains, providing a motion which is 
      critical for orienting the partners involved in the hydride transfer reaction. It 
      has previously been proposed that this conformational change is triggered by 
      substrate binding. However, analysis of the K89L mutant shows that it adopts an 
      almost identical conformation with that of the wild-type enzyme in the presence 
      of substrate. Comparison of the mutant structure with both the wild-type open and 
      closed forms has enabled us to separate conformational changes associated with 
      substrate binding and domain motion and suggests that the domain closure may well 
      be a property of the wild-type enzyme even in the absence of substrate.
CI  - Copyright 1999 Academic Press.
FAU - Stillman, T J
AU  - Stillman TJ
AD  - University of Sheffield, Sheffield, Western Bank, S10 2TN, UK.
FAU - Migueis, A M
AU  - Migueis AM
FAU - Wang, X G
AU  - Wang XG
FAU - Baker, P J
AU  - Baker PJ
FAU - Britton, K L
AU  - Britton KL
FAU - Engel, P C
AU  - Engel PC
FAU - Rice, D W
AU  - Rice DW
LA  - eng
SI  - PDB/1K89
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - EC 1.4.- (Amino Acid Oxidoreductases)
RN  - EC 1.4.1.2 (Glutamate Dehydrogenase)
RN  - EC 1.4.1.9 (Leucine Dehydrogenase)
RN  - GMW67QNF9C (Leucine)
RN  - K3Z4F929H6 (Lysine)
SB  - IM
MH  - Amino Acid Oxidoreductases/metabolism
MH  - Amino Acid Sequence
MH  - Clostridium/*enzymology
MH  - Glutamate Dehydrogenase/*chemistry/genetics/*metabolism
MH  - Leucine/genetics/*metabolism
MH  - Leucine Dehydrogenase
MH  - Lysine/genetics/*metabolism
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Protein Conformation
MH  - Structure-Activity Relationship
MH  - Substrate Specificity
EDAT- 1999/01/08 00:00
MHDA- 1999/01/08 00:01
CRDT- 1999/01/08 00:00
PHST- 1999/01/08 00:00 [pubmed]
PHST- 1999/01/08 00:01 [medline]
PHST- 1999/01/08 00:00 [entrez]
AID - S0022-2836(98)92335-X [pii]
AID - 10.1006/jmbi.1998.2335 [doi]
PST - ppublish
SO  - J Mol Biol. 1999 Jan 15;285(2):875-85. doi: 10.1006/jmbi.1998.2335.