Biomedical subjects

William S McIntire

Publications and source records attributed to William S McIntire.

7 recordsLinked to original sources

PubMed2005

p-Cresol methylhydroxylase: alteration of the structure of the flavoprotein subunit upon its binding to the cytochrome subunit.

The structures of two forms of a recombinant flavoprotein have been determined at high resolution and compared. These proteins are (1) the flavocytochrome c p-cresol methylhydroxylase (rPCMH, 1.85 A resolution) and (2) the cytochrome-free flavoprotein subunit of rPCMH (PchF, 1.30 A resolution). A significant conformational difference is observed in a protein segment that is in contact with the re face of the isoalloxazine ring of FAD when the structure of PchF is compared to the subunit in the intact flavocytochrome. This structural change is important for optimum catalytic function of the flavoprotein, which has been shown to be dependent on the presence of the cytochrome subunit. This change results in different protein-flavin and apparently different protein-substrate interactions that have a "tuning effect" on the electronic and redox properties of bound p-cresol and the covalently bound FAD. The conformational change in the segment in the cofactor-binding site is induced by a small rearrangement in the flavoprotein-cytochrome interface region of the flavoprotein.

Amino Acid Sequence↗

PubMed2005

Relationship between charge-transfer interactions, redox potentials, and catalysis for different forms of the flavoprotein component of p-cresol methylhydroxylase.

Thirty-three variants of the flavoprotein component of p-cresol methylhydroxylase that contain noncovalently or covalently bound flavin adenine dinucleotide (FAD) analogues were studied. A very good correlation was found between the efficiency of p-cresol oxidation by these proteins and E(CT), the energy for the maximum wavelength for the charge-transfer band of the complex between the bound flavin and 4-bromophenol, a substrate mimic. The correlation covers a range of k(cat) values that spans over 5 orders of magnitude and values of E(CT) that span 900 mV, and the analysis of the data provided a value of the transfer coefficient, alpha, of 0.31. This study demonstrates clearly that the redox properties of both the bound substrate and the flavin cofactor must be taken into account to explain the relative catalytic efficiencies of the variant flavoproteins.

Catalysis↗

PubMed2005

Inhibition of murine N1-acetylated polyamine oxidase by an acetylenic amine and the allenic amine, MDL 72527.

Murine N(1)-acetylated polyamine oxidase (mPAO) was treated with N,N'-bis-(prop-2-ynyl)-1,4-diaminobutane, a poor substrate and inhibitor for the enzyme, with K(m) and K(i) values in the millimolar range. Apparently, its oxidation produces prop-2-ynal, which reacts with amino acyl nucleophiles. Using a steady-state kinetic assay, four phases were identified, the first being the oxidation of the compound via Michealis-Menten-type kinetics. As prop-2-ynal accumulates, there is a biphasic reduction in the rate. This process leads to an mPAO form that is nearly inactive (fourth phase), but displays classical Michealis-Menten-type kinetics. The enzyme-bound flavin is not modified in this process. In contrast, micromolar concentrations of the MDL 72527 (N,N'-bis-[buta-2,3-dienyl]-1,4-diaminobutane) inhibited mPAO rapidly and completely. It inhibits by first binding tightly and apparently irreversibly, and then slowly converts to a species where the inhibitor is covalently bound to the N5-position of the flavin's isoalloxazine ring. The covalent adduct was identified as a flavocyanine.

Acetylation↗

PubMed2004

Oxygen isotope effects on electron transfer to O2 probed using chemically modified flavins bound to glucose oxidase.

Apo-glucose oxidase has been reconstituted with flavins modified in the 7 and 8 positions and characterized with regard to the catalytic rate of O(2) reduction and oxygen-18 isotope effects on this process. Kinetic studies as a function of driving force indicate a reorganization energy for electron transfer to O(2) of lambda = 28 kcal mol(-)(1) at optimal pH, which is similar to the value obtained earlier from temperature dependencies of rates (Roth, J. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 62-67). For the various enzyme-bound flavins, competitive oxygen-18 kinetic isotope effects fall within the narrow range of 1.0266(5) to 1.0279(6), apparently because of the dominant contribution of outer-sphere reorganization to the activation barrier; within the context of semiclassical and quantum mechanical electron transfer theories, the magnitude of the isotope effects reveals the importance of nuclear tunneling.

Aspergillus niger↗

PubMed2004

A study of the spectral and redox properties and covalent flavinylation of the flavoprotein component of p-cresol methylhydroxylase reconstituted with FAD analogues.

The spectral and redox properties are described for the wild-type and Y384F mutant forms of the flavoprotein component (PchF) of flavocytochrome, p-cresol methylhydroxylase (PCMH), and cytochrome-free PchF that harbor FAD analogues. The analogues are iso-FAD (8-demethyl-6-methyl-FAD), 6-amino-FAD (6-NH(2)-FAD), 6-bromo-FAD (6-Br-FAD), 8-nor-8-chloro-FAD (8-Cl-FAD), and 5-deaza-5-carba-FAD (5-deaza-FAD). All of the analogues bound noncovalently and stoichiometrically to cytochrome-free apo-PchF, and the resulting holoproteins had high affinity for the cytochrome subunit, PchC. Noncovalently bound FAD, 6-Br-FAD, or 6-NH(2)-FAD can be induced to bind covalently by exposing holo-PchF to PchC. The rate of this process and the redox potential of the noncovalently bound flavin may be correlated. In addition, the redox potential of each FAD analogue was higher when it was covalently bound than when noncovalently bound to PchF. Furthermore, the potential of a covalently bound or noncovalently bound FAD analogue increased on association of the corresponding holo-PchF with PchC, and the activity increased as the flavin's redox potential increased. It was discovered also that 4-hydroxybenzaldehyde, the final p-cresol oxidation product, is an efficient competitive inhibitor for substrate oxidation by PchF since it binds tightly to this protein when the flavin is oxidized, although it binds more loosely to the enzyme with reduced flavin. Finally, the energies of the charge-transfer bands for the interaction of bound flavin analogues with 4-Br-phenol (a substrate mimic) increased as the potential decreases, although a simple global correlation was not seen. This is the case because the energy is also a function of the redox properties of the bound mimic. The implications of these findings to covalent flavinylation and catalysis are discussed.

Binding Sites↗

PubMed2004

Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.

Each flavoprotein subunit (PchF) of p-cresol methylhydroxylase (PCMH) has flavin adenine dinucleotide (FAD) covalently tethered to Tyr384. The PCMH structure suggests that Arg474 in PchF is required for self-catalytic covalent flavinylation and for substrate oxidation. The replacement of Arg474 with Lys was carried out to probe the subtleties of the role of Arg474 in these processes. In nearly all of the aspects examined, the mutant protein showed compromised properties relative to the wild-type protein, including the tenacity of noncovalent FAD binding to the apo-protein, the rate of covalent flavinylation, the affinity of the covalent flavoprotein for PchC (the cytochrome subunit), the k(cat) for substrate oxidation, and the affinity for substrate analogues in the formation of FAD-charge-transfer complexes (CT complexes). Nevertheless, because the mutant retains these attributes, the comparison allows for an examination of the role of this residue in the various properties of the enzyme. A correlation is proposed to exist between nu(m), the frequency for the absorbance maximum of the CT complex with a substrate analogue, and k(cat), the steady-state rate constant for oxidation of p-cresol by various forms of PCMH and PchF; both nu(m) and k(cat) can be expressed as functions of the ionization potential of the donor (I(D)) and the electron affinity of the acceptor (E(A)). This correlation is a better predictor of the rate constant for substrate oxidation than is the magnitude of the redox potential, E(m,7), of the bound FAD, which was determined for the various mutant enzyme species and compared with those of the wild type.

Animals↗

PubMed2003

Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase.

The aminoacyl sequences of three regions of pure bovine N1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBankTM. This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBankTM indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a -Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N1-acetylated polyamines on the exo-side of their N4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine (but not N1-acetylspermine or N1-acetylspermidine) at the exo-carbon of its N4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.

Amino Acid Sequence↗