Biomedical subjects

F C Hartman

Publications and source records attributed to F C Hartman.

At least 91 records · Page 5Linked to original sources

PubMed1980

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase. Sequence homology with triosephosphate isomerase.

N-Bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pKa values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivty methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

Affinity Labels↗

PubMed1979

C nuclear magnetic resonance study of the CO(2) activation of ribulosebisphosphate carboxylase from Rhodospirillum rubrum.

Ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from Rhodospirillum rubrum is activated by CO(2) and Mg(2+). (13)C NMR spectra were determined for the unactivated enzyme and for enzyme that had been activated by (13)CO(2) and Mg(2+). In addition to the expected resonance for H(13)CO(3) (-)/CO(3) (2-) at 161.8 ppm downfield from tetramethylsilane, the spectrum of the activated enzyme shows a broad resonance at 164.9 ppm. Analogy with previous NMR studies of (13)CO(2) binding to hemoglobin [Morrow, J. S., Keim, P., Visscher, R. B., Marshall, R. C. & Gurd, F. R. N. (1973) Proc. Natl. Acad. Sci. USA 70, 1414-1418], to myoglobin, and to amino acids [Morrow, J. S., Keim, P. & Gurd, F. R. N. (1974) J. Biol. Chem. 249, 7484-7494] suggests that the CO(2) activation of ribulosebisphosphate carboxylase involves formation of a carbamate between an enzyme amino group and CO(2).

Journal Article↗

PubMed1979

Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum.

Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.

Amino Acids↗

PubMed1978

Inactivation of ribulosebisphosphate carboxylase by modification of arginyl residues with phenylglyoxal.

Phenylglyoxal rapidly and completely inactivates spinach and Rhodospirillum rubrum ribulosebisphosphate carboxylases. Inactivation exhibits pseudo-first-order kinetics and a reaction order of approximately one for both enzymes, suggesting that modification of a single residue per protomeric unit suffices for inactivation. Loss of enzymic activity is directly proportional to incorporation of [14C]phenylglyoxal until only 30% of the initial activity remains. For both enzymes, extrapolation of incorporation to 100% inactivation yields 4-5 mol of [14C]phenylglyoxal per mol protomer. Amino acid analyses confirm the expected 2:1 stoichiometry between phenylglyoxal incorporation and arginyl modification and suggest that other kinds of amino acid residues are not modified. (Thus, inactivation correlates with modification of 2-3 arginyl residues per protomer). The substrate ribulose bis-phosphate and some competitive inhibitors reduce the rates of inactivation of the carboxylases and prevent modification of about 0.5-1.0 arginyl residue per protomer. Inactivation is therefore a consequence of modification of a small number of residues out of the 35 and 29 total arginyl residues per protomer in spinach and R. rubrum carboxylases, respectively.

Aldehydes↗

PubMed1978

Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate.

We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).

Affinity Labels↗

PubMed1978