Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.
Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.
Warren
M J
MJ
Department of Medical Genetics, University of London, U.K.
Gul
S
S
Aplin
R T
RT
Scott
A I
AI
Roessner
C A
CA
O'Grady
P
P
Shoolingin-Jordan
P M
PM
D000478:Alkylation; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D011163:Hydroxymethylbilane Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation
D011163:Hydroxymethylbilane Synthase; D003545:Cysteine; D005033:Ethylmaleimide