Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase.
The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.
Scott
A I
AI
Chemistry Department, Texas A&M University, College Station 77843-3255.
Roessner
C A
CA
Stolowich
N J
NJ
Karuso
P
P
Williams
H J
HJ
Grant
S K
SK
Gonzalez
M D
MD
Hoshino
T
T
D000642:Ammonia-Lyases / Q000378:metabolism*; D001665:Binding Sites; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D010957:Plasmids
D002247:Carbon Isotopes; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases