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

Abstract

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.more » A mechanistic rationale for deaminase is presented.« less

Authors:
; ; ; ; ; ; ;  [1]
  1. Texas A M Univ., College Station (USA)
Publication Date:
OSTI Identifier:
6976534
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 27:21; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Scott, A I, Roessner, C A, Stolowich, N J, Karuso, P, Williams, H J, Grant, S K, Gonzalez, M D, and Hoshino, T. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase. United States: N. p., 1988. Web. doi:10.1021/bi00421a002.
Scott, A I, Roessner, C A, Stolowich, N J, Karuso, P, Williams, H J, Grant, S K, Gonzalez, M D, & Hoshino, T. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase. United States. https://doi.org/10.1021/bi00421a002
Scott, A I, Roessner, C A, Stolowich, N J, Karuso, P, Williams, H J, Grant, S K, Gonzalez, M D, and Hoshino, T. 1988. "Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase". United States. https://doi.org/10.1021/bi00421a002.
@article{osti_6976534,
title = {Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase},
author = {Scott, A I and Roessner, C A and Stolowich, N J and Karuso, P and Williams, H J and Grant, S K and Gonzalez, M D and Hoshino, T},
abstractNote = {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.},
doi = {10.1021/bi00421a002},
url = {https://www.osti.gov/biblio/6976534}, journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 27:21,
place = {United States},
year = {Tue Oct 18 00:00:00 EDT 1988},
month = {Tue Oct 18 00:00:00 EDT 1988}
}