title: The Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG): autocatalytic formation and effect on enzyme catalysis and spectroscopic properties.
abstract: Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, 94143, USA.
authors.forename: Reza A
authors.initials: RA
authors.lastname: Ghiladi
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Knudsen
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry / Q000502:physiology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D010463:Peracetic Acid / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D014364:Tryptophan; D008715:Methionine; D006861:Hydrogen Peroxide; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D014357:Trypsin; D010463:Peracetic Acid