PMID- 22855027
OWN - NLM
STAT- MEDLINE
DCOM- 20130304
LR  - 20211021
IS  - 1469-896X (Electronic)
IS  - 0961-8368 (Print)
IS  - 0961-8368 (Linking)
VI  - 21
IP  - 11
DP  - 2012 Nov
TI  - Exploration of the active site of Escherichia coli cystathionine gamma-synthase.
PG  - 1662-71
LID - 10.1002/pro.2135 [doi]
AB  - Cystathionine gamma-synthase (CGS) catalyzes the condensation of 
      O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce 
      L-cystathionine (L-Cth) and succinate, in the first step of the bacterial 
      transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the 
      futile alpha,gamma-elimination of L-OSHS, yielding succinate, alpha-ketobutyrate, and 
      ammonia. A series of 16 site-directed variants of Escherichia coli CGS (eCGS) was 
      constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, 
      R106, N227, E325, S326, and R361. The effects of these substitutions on the 
      catalytic efficiency of the alpha,gamma-elimination reaction range from a reduction of 
      only  approximately 2-fold for R49K and the E325A,Q variants to 310- and 760-fold for R361K and 
      R48K, respectively. A similar trend is observed for the k(cat) /K(m)(l-OSHS) of 
      the physiological, alpha,gamma-replacement reaction. The results of this study suggest 
      that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in 
      bacterial CGS sequences, tether the distal and alpha-carboxylate moieties, 
      respectively, of the L-OSHS substrate. In contrast, with the exception of the 
      13-fold increase observed for R106A, the K(m)(l-Cys) is not markedly affected by 
      the site-directed replacement of the residues investigated. The decrease in 
      k(cat) observed for the S326A variant reflects the role of this residue in 
      tethering the side chain of K198, the catalytic base. Although no structures 
      exist of eCGS bound to active-site ligands, the roles of individual residues is 
      consistent with the structures inhibitor complexes of related enzymes. 
      Substitution of D45, E325, or Y101 enables a minor transamination activity for 
      the substrate L-Ala.
CI  - Copyright (c) 2012 The Protein Society.
FAU - Jaworski, Allison F
AU  - Jaworski AF
AD  - Department of Biology, Carleton University, Ottawa K1S 5B6, Canada.
FAU - Lodha, Pratik H
AU  - Lodha PH
FAU - Manders, Adrienne L
AU  - Manders AL
FAU - Aitken, Susan M
AU  - Aitken SM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Protein Sci
JT  - Protein science : a publication of the Protein Society
JID - 9211750
RN  - 0 (Escherichia coli Proteins)
RN  - 375YFJ481O (Cystathionine)
RN  - 5V5IOJ8338 (Pyridoxal Phosphate)
RN  - EC 2.5.1.48 (O-succinylhomoserine (thiol)-lyase)
RN  - EC 4.2.- (Carbon-Oxygen Lyases)
SB  - IM
MH  - Carbon-Oxygen Lyases/chemistry/genetics/*metabolism
MH  - Catalytic Domain
MH  - Cystathionine/chemistry/metabolism
MH  - Escherichia coli/*enzymology/genetics
MH  - Escherichia coli Proteins/chemistry/genetics/metabolism
MH  - Kinetics
MH  - Models, Molecular
MH  - Mutagenesis, Site-Directed
MH  - Mutation
MH  - Pyridoxal Phosphate/chemistry/metabolism
PMC - PMC3527702
EDAT- 2012/08/03 06:00
MHDA- 2013/03/05 06:00
PMCR- 2013/11/01
CRDT- 2012/08/03 06:00
PHST- 2012/08/03 06:00 [entrez]
PHST- 2012/08/03 06:00 [pubmed]
PHST- 2013/03/05 06:00 [medline]
PHST- 2013/11/01 00:00 [pmc-release]
AID - 10.1002/pro.2135 [doi]
PST - ppublish
SO  - Protein Sci. 2012 Nov;21(11):1662-71. doi: 10.1002/pro.2135.