PMID- 21958132
OWN - NLM
STAT- MEDLINE
DCOM- 20120103
LR  - 20111108
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Linking)
VI  - 50
IP  - 45
DP  - 2011 Nov 15
TI  - Characterization of the side-chain hydroxyl moieties of residues Y56, Y111, Y238, 
      Y338, and S339 as determinants of specificity in E. coli cystathionine beta-lyase.
PG  - 9876-85
LID - 10.1021/bi201090n [doi]
AB  - Cystathionine beta-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) 
      to produce L-homocysteine, pyruvate, and ammonia. A series of site-directed 
      variants of Escherichia coli CBL (eCBL) was constructed to investigate the roles 
      of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 
      as determinants of specificity. The effect of these conservative substitutions on 
      the k(cat)/K(m)(L-Cth) for the alpha,beta-elimination of L-Cth ranges from a change of 
      only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine 
      replacement variant of S339. A novel role for residue S339 as a determinant of 
      reaction specificity, via tethering of the catalytic base, K210, is demonstrated. 
      Comparison of the kinetic parameters for L-Cth hydrolysis with those for the 
      inhibition of eCBL by aminoethoxyvinylglycine (AVG) indicates that Y238 interacts 
      with the distal carboxylate group of the substrate. The 22 and 50-fold increases 
      in the K(m)(L-Cth) and K(i)(AVG) resulting from replacement of Y56 with 
      phenylalanine suggest that this residue may interact with the distal amino group 
      of these compounds, although an indirect role in binding is more likely. The 
      near-native k(cat)/K(m)(L-Cth) and pH profile of the eCBL-Y111F variant 
      demonstrate that residue Y111 does not play a role in proton transfer. The 
      understanding of the eCBL active site and of the determinants of substrate and 
      reaction specificity resulting from this work will facilitate the design of 
      inhibitors, as antibacterial therapeutics, and the engineering of enzymes 
      dependent on the catalytically versatile pyridoxal 5'-phosphate cofactor to 
      modify reaction specificity.
FAU - Lodha, Pratik H
AU  - Lodha PH
AD  - Department of Biology, Carleton University, Ottawa, Canada K1S 5B6.
FAU - Aitken, Susan M
AU  - Aitken SM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20111025
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (Recombinant Proteins)
RN  - EC 4.- (Lyases)
RN  - EC 4.4.1.8 (cystathionine beta-lyase)
SB  - IM
MH  - Amino Acid Substitution
MH  - Catalytic Domain/genetics
MH  - Escherichia coli/enzymology/genetics
MH  - Escherichia coli Proteins/*chemistry/genetics/*metabolism
MH  - Hydrogen-Ion Concentration
MH  - Kinetics
MH  - Lyases/*chemistry/genetics/*metabolism
MH  - Models, Molecular
MH  - Mutagenesis, Site-Directed
MH  - Recombinant Proteins/chemistry/genetics/metabolism
MH  - Substrate Specificity
EDAT- 2011/10/01 06:00
MHDA- 2012/01/04 06:00
CRDT- 2011/10/01 06:00
PHST- 2011/10/01 06:00 [entrez]
PHST- 2011/10/01 06:00 [pubmed]
PHST- 2012/01/04 06:00 [medline]
AID - 10.1021/bi201090n [doi]
PST - ppublish
SO  - Biochemistry. 2011 Nov 15;50(45):9876-85. doi: 10.1021/bi201090n. Epub 2011 Oct 
      25.