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F C Hartman

Publications and source records attributed to F C Hartman.

At least 73 records · Page 4Linked to original sources
PubMed1985

Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase.

Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.

Amino Acid Sequence↗
PubMed1984

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.

Adenosine Triphosphate↗
PubMed1984

Preliminary structural studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Rhodospirillum rubrum has been crystallized in a form that is suitable for structural studies by x-ray diffraction. The asymmetric unit of the crystal contains one dimeric enzyme molecule of molecular mass 101,000 Da. The enzyme was activated prior to crystallization and is presumed to be in the CO2-activated state in the crystal. The method of hydrophobicity correlation has been used to compare the amino acid sequence of this molecule (466 residues) to that of the large subunit of a higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (477 residues in Nicotiana tabacum). The pattern of residue hydrophobicities is similar along the two polypeptides. This suggests that the three-dimensional folding of the large polypeptide chains may be similar in plant and bacterial enzymes. If this is so, knowing the structure of either the plant or bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase should aid in learning the structure of the other.

Amino Acid Sequence↗
PubMed1984

Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction.

At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.

Borohydrides↗
PubMed1984

2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide.

A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus of the enzyme. The lack of reactivity with the spinach enzyme is explained by the deletion of the histidyl residue and the replacement of cysteine by tryptophan in the eukaryotic species. Although the nonconservation of the modified residues argues against a functional role other than maintenance of structural integrity, the extensive homology in this region among seven different species of carboxylase is compatible with the region comprising a portion of the active site.

Affinity Labels↗
PubMed1984

The shape of ribulose bisphosphate carboxylase/oxygenase in solution as inferred from small angle neutron scattering.

Small angle neutron scattering of both activated and deactivated hexadecameric ribulose bisphosphate carboxylase/oxygenase from spinach revealed that its structure in solution very closely resembles that determined for the deactivated crystalline enzyme from tobacco (Baker, T.S., Eisenberg, D., and Eiserling, F. (1977) Science 196, 293-295). Scattering from both forms of the enzyme in H2O most closely fits that expected from a hollow sphere with an outer radius of 56.4 A and inner radius of 14.3 A. In D2O the enzyme's scattering profiles more closely resembled those of a hollow cylinder with an axial ratio of approximately 1.06 and outer and inner radii of 46.7 and 12.9 A, respectively. Superpositioning of model structures showed that both models determined by neutron scattering correspond quite well to that inferred from x-ray diffraction and electron microscopy of the tobacco enzyme. Sedimentation velocity studies confirmed that no major conformational change occurs upon activation of the spinch carboxylase, both forms exhibiting a sedimentation coefficient, 8(20,w), of 17.8 S. Neutron scattering by activated and deactivated carboxylase from Rhodospirillum rubrum, a dimeric enzyme, best matched that predicted for a solid prolate ellipsoid or cylinder. The estimated dimensions of the model structures correspond strikingly well to those predicted for adjacent pairs of large subunits in the hexadecameric tobacco enzyme.

Molecular Weight↗
PubMed1984

Complete primary structure of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.

Amino Acid Sequence↗
PubMed1983

Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.

2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.

Affinity Labels↗
PubMed1982

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.

Amino Acids↗
PubMed1982

Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed in contrast to an earlier report [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. Subsequent to reduction with [3H]borohydride and tryptic digestion of the enzyme inactivated by PLP, the sole labeled peptide was purified by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. The peptide, recovered in good yield, appeared essentially homogeneous by amino acid analysis, peptide mapping, and sequencing. Automated Edman degradation established the peptide's sequence as Val-Leu-Gly-Arg-Pro-Glu-Val-Asp-Gly-Gly-Leu-Val-Val-Gly-Thr-Ile-Ile-(PLP)Lys -Pro-Lys instead of Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP)Lys-Gly-Thr-Leu-Val-Ile-Lys as reported by Robison et al. (1980) [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. The sequence -Ile-Lys-Pro-Lys- in the former is identical with that encompassing Lys-175 in the carboxylase/oxygenase from spinach, which reacts preferentially with PLP and two other affinity labels. This finding of homology greatly strengthens the supposition that Lys-175 in the spinach enzyme and the corresponding lysyl residue in the Rhodospirillum rubrum enzyme are active-site residues and furthermore increases the likelihood of their functionality in catalysis.

Amino Acid Sequence↗
PubMed1981

Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for alpha-ketoglutarate binding sites.

The interaction of oxidized nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for alpha-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentrations of regent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and Kinact for bromoketoglutarate is 250 microM. Isocitrate and NADP+ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (Ki = 100 microM) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a Km of 250 microM and a Vmax comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (Ki = 3 microM) and ketoglutarate (Ki = 50 microM). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO2 is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed. Commercial isocitrate dehydrogenase is purified 12-fold by affinity chromatography on thiol-agarose alkylated by bromoketoglutarate.

Affinity Labels↗