Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

Journal Article · Tue Oct 18 00:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00421a002· OSTI ID:6976534

Scott, A I; Roessner, C A; Stolowich, N J; Karuso, P; Williams, H J; Grant, S K; Gonzalez, M D; Hoshino, T ^[1]

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1. Texas A M Univ., College Station (USA)

The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-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-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} 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.

• OSTI ID: 6976534
• Journal Information: Biochemistry; (USA), Vol. 27:21; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
ENZYME INHIBITORS
CROSS-LINKING
LYASES
NUCLEAR MAGNETIC RESONANCE
AMINOLEVULINIC ACID
CARBON 13
CYSTEINE
ELECTROPHORESIS
ESCHERICHIA COLI
MOLECULAR STRUCTURE
MUTAGENESIS
NMR SPECTRA
PORPHYRINS
PROTONS
SERINE
SUBSTRATES
AMINO ACIDS
BACTERIA
BARYONS
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ELEMENTARY PARTICLES
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
POLYMERIZATION
RESONANCE
SPECTRA
STABLE ISOTOPES
THIOLS
550601* - Medicine- Unsealed Radionuclides in Diagnostics