PMID- 1521535
OWN - NLM
STAT- MEDLINE
DCOM- 19921013
LR  - 20190620
IS  - 0014-2956 (Print)
IS  - 0014-2956 (Linking)
VI  - 208
IP  - 2
DP  - 1992 Sep 1
TI  - Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from 
      Escherichia coli.
PG  - 443-9
AB  - The anomeric specificity and the steady-state kinetic mechanism of homogeneous 
      3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. 
      The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is 
      structurally prohibited from undergoing ring closure, was synthesized and tested 
      as a substrate for the synthase. It was found that the analogue functions as a 
      substrate with a similar kcat value to that of the original substrate. The 
      kcat/Km value for the natural substrate is seven-times greater than that of the 
      4-deoxy analogue. However, taking into account the 9.5% and approximately 1% 
      concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in 
      solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 
      microM, respectively, requiring about a 3 kcal/mol contribution to the binding 
      energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the 
      enzyme acts upon the acyclic form of the natural substrate. The steady-state 
      kinetic study of KDO8P synthase was analyzed via inhibition using the products 
      KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. 
      First, intersecting lines in double-reciprocal plots of initial-velocity data at 
      substrate concentrations in the micromolar range suggest a sequential mechanism 
      for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is 
      competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). 
      These inhibition patterns are consistent with the model wherein P-pyruvate 
      binding precedes that of Ara5P binding. Furthermore, this order of substrate 
      binding was supported by the observations that KDO8P is a competitive inhibitor 
      for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to 
      the same enzyme form, and noncompetitively with respect to Ara5P. In addition, 
      the inhibition by inorganic phosphate is noncompetitive with respect to both 
      P-pyruvate and Ara5P, suggesting an apparent ordered release of products such 
      that Pi first, followed by KDO8P. In conclusion, these data suggest a 
      steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding 
      precedes that of Ara5P, followed by the ordered release of inorganic phosphate 
      and KDO8P.
FAU - Kohen, A
AU  - Kohen A
AD  - Department of Chemistry, Technion-Israel Institute of Technology, Haifa.
FAU - Jakob, A
AU  - Jakob A
FAU - Baasov, T
AU  - Baasov T
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - England
TA  - Eur J Biochem
JT  - European journal of biochemistry
JID - 0107600
RN  - 0 (Phosphates)
RN  - 0 (Ribosemonophosphates)
RN  - 4B2428FLTO (ribose-5-phosphate)
RN  - 73-89-2 (Phosphoenolpyruvate)
RN  - B40ROO395Z (Arabinose)
RN  - EC 2.5.1.55 (2-dehydro-3-deoxyphosphooctonate aldolase)
RN  - EC 4.1.2.- (Aldehyde-Lyases)
SB  - IM
MH  - Aldehyde-Lyases/antagonists & inhibitors/*metabolism
MH  - Arabinose/analogs & derivatives/chemistry/metabolism
MH  - Binding, Competitive
MH  - Escherichia coli/*enzymology
MH  - Kinetics
MH  - Phosphates/chemical synthesis/metabolism
MH  - Phosphoenolpyruvate/metabolism
MH  - Ribosemonophosphates/pharmacology
MH  - Substrate Specificity
EDAT- 1992/09/01 00:00
MHDA- 1992/09/01 00:01
CRDT- 1992/09/01 00:00
PHST- 1992/09/01 00:00 [pubmed]
PHST- 1992/09/01 00:01 [medline]
PHST- 1992/09/01 00:00 [entrez]
AID - 10.1111/j.1432-1033.1992.tb17206.x [doi]
PST - ppublish
SO  - Eur J Biochem. 1992 Sep 1;208(2):443-9. doi: 10.1111/j.1432-1033.1992.tb17206.x.