Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.
BACKGROUND
Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion.


RESULTS
With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior.


CONCLUSIONS
Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.
Jarret
C
C
Institute of Chemistry, University of Neuchâtel, Neuchâtel, CH-2000, Switzerland.
Stauffer
F
F
Henz
M E
ME
Marty
M
M
Lüönd
R M
RM
Bobálová
J
J
Schürmann
P
P
Neier
R
R
D000622:Aminolevulinic Acid / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D020134:Catalytic Domain; D003998:Dicarboxylic Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D011162:Porphobilinogen / Q000737:chemistry / Q000378:metabolism; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D011162:Porphobilinogen; D000622:Aminolevulinic Acid; D000623:Porphobilinogen Synthase