Biomedical subjects

D B Goodin

Publications and source records attributed to D B Goodin.

6 recordsLinked to original sources

PubMed1991

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1989

Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.

The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H2O2-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century. Site-directed mutagenesis alone cannot yield this answer. Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp51----Phe. The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp191.

Binding Sites↗

PubMed1988

A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligodeoxynucleotides.

A very simple and highly efficient procedure for the generation of single and multiple substitutions in segments of DNA is described which has no requirements for conveniently placed restriction sites, and allows all DNA sequences to be equally accessible. A mixed pool of oligodeoxynucleotides is synthesized by contaminating the monomeric nucleotides with low levels of the other three nucleotides such that the full-length oligonucleotide contains on the average one to two changes per molecule. This pool is used in priming in vitro synthesis of the complementary strand of cloned DNA fragments in M13 or pEMBL vectors which have previously been passed through a dut-, ung- Escherichia coli host. Strong selection for the newly synthesized strand is provided by transforming the heteroduplex into a dut+, ung+ host. Single and multiple substitutions in the carboxy-terminal coding region of the MATa1 gene of Saccharomyces cerevisiae are introduced at high efficiency (25-55%) and the changes are identified by direct sequencing alone. The same principle can be used to generate multiple sets of changes at any specified codon.

Codon↗

PubMed1988

A rapid droplet method for Sanger dideoxy sequencing.

A method for performing the dideoxy sequence reaction on petri dishes is described. It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

Base Sequence↗

PubMed1987

The peroxide complex of yeast cytochrome c peroxidase contains two distinct radical species, neither of which resides at methionine 172 or tryptophan 51.

The nature of the free radical species observed in the peroxide complex of yeast cytochrome c peroxidase is described for protein variants containing amino acid substitutions at Met-172 and Trp-51. As was the case with Met-172 mutations (Goodin, D.B., Mauk, A.G., and Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299), Trp-51 can be substituted to give active enzyme. Phe-51-containing enzyme has a higher turnover rate than the original enzyme and exhibits an altered pH dependence. The properties of the isotropic and axial components (Hoffman, B.M., Roberts, J.E., Kang, C.H., and Margoliash, E. (1981) J. Biol. Chem. 256, 6556-6564; Hori, H., and Yonetani, T. (1985) J. Biol. Chem. 260, 349-355) of the EPR signal of the wild-type enzyme-peroxide complex, studied as a function of H2O2 stoichiometry, support proposals (Goodin et al. (1985) and Hori and Yonetani (1985), see above) that two distinct radical species are formed, and spin quantification shows that the isotropic radical is always formed in substoichiometric amounts. The peroxide complexes for proteins containing amino acid substitutions at either Met-172 or Trp-51 exhibit somewhat larger than normal levels of the isotropic radical signal. In addition, these mutants are unlike wild-type enzyme in that the axial EPR signal associated with the peroxide complex is seen only at 10 K and not at 90 K. Thus, neither amino acid can be considered to be the molecular species responsible for either radical signal, but both mutations appear to affect the physical properties of the axial signal representing the major radical species.

Cloning, Molecular↗

PubMed1986

Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.

Amino Acid Sequence↗