Aspartate Transcarbamylase from Escherichia coli

Greenwell, P.; Jewett, Sandra L.; Stark, George R.

doi:10.1016/s0021-9258(19)43498-4

Cited by 86 publications

(37 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An erroneous interpretation of the fluorescence quenching can result if there is nonspecific binding of the donor or acceptor molecules. The site specificity of the pyridoxamine phosphate labeling has been demonstrated through inhibition studies, spectral measurements, and amino acid analysis (Greenwell et al, 1973). The stoichiometric binding and specific location of mercurinitrophenol have been demonstrated in titration and X-ray diffraction studies (Evans et al, 1972).…”

Section: Discussionmentioning

confidence: 99%

“…Protein concentrations were determined from the absorbance at 280 nm using extinction coefficients of 0.59 ml/(mg cm) for the native enzyme, 0.72 ml/(mg cm) for the unmodified catalytic subunit, and 0.32 ml/(mg cm) for the zinc regulatory subunit (Gerhart and Holoubek, 1967;Nelbach et al, 1972). Pyridoxamine phosphate conjugates of the catalytic subunit were prepared by sodium borohydride reduction of the Schiff base formed between pyridoxal phosphate and an amino group on the protein (Greenwell et al, 1973). The extent of labeling was determined from the absorbance at 325 nm using a molar extinction coefficient of 5350 M-1 cm-1 after correction for protein absorbance at that wavelength (Greenwell et al, 1973).…”

Section: Methodsmentioning

confidence: 99%

“…Pyridoxamine phosphate conjugates of the catalytic subunit were prepared by sodium borohydride reduction of the Schiff base formed between pyridoxal phosphate and an amino group on the protein (Greenwell et al, 1973). The extent of labeling was determined from the absorbance at 325 nm using a molar extinction coefficient of 5350 M-1 cm-1 after correction for protein absorbance at that wavelength (Greenwell et al, 1973). Several derivatives were made containing a range of 1.6-3.2 mol of pyridoxamine phosphate/mol of catalytic trimer.…”

Section: Methodsmentioning

confidence: 99%

“…Renatured enzyme labeled with pyridoxamine phosphate was made by mixing catalytic subunit derivatives labeled with approximately 3 mol of pyridoxamine phosphate/mol of trimer with excess (20% with respect to polypeptide chains) zinc-regulatory subunit. The mixture of subunits at protein concentrations of about 1 mg/ml in 0.05 M Tris-acetate buffer (pH 8) with 2 mM mercaptoethanol and 0.1 mM zinc acetate was incubated for 30 min at 30° ( Greenwell et al, 1973). After precipitation with ammonium sulfate and dialysis against 0.05 M Tris-acetate buffer (pH 8) containing 2 mM mercaptoethanol and 0.2 mM EDTA, the enzyme solution was chromatographed on a 3 cm i.d.…”

Section: Methodsmentioning

confidence: 99%

“…Weber, 1968a; Stryer, 1968;Yguerabide, 1972). Two sitespecific chromophores have been attached to aspartate transcarbamylase: pyridoxamine phosphate at the catalytic site (Greenwell et al, 1973) and mercurinitrophenol at the sulfhydryl groups on the catalytic subunit (Evans et al, 1972). In this study fluorescence energy transfer between these and other labels is used to measure the distances from a catalytic site to another catalytic site, to the tryptophan polypeptide residues, to adjacent and nonadjacent sulfhydryl groups and to the nucleotide binding site.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase

Matsumoto¹,

Hammes²

1975

Biochemistry

View full text Add to dashboard Cite

The method of fluorescence energy transfer is used to measure the distances between several sites on aspartate transcarbamylase. Both fluorescence steady-state and lifetime techniques are used. When the tryptophans on the catalytic subunit are the fluorescent donor groups, either pyridoxamine phosphate, covalently bound to an amino group at the active site, or 8-anilino-1-naphthalenesulfonate, noncovalently bound at the active site, is the acceptor group. The distance between tryptophan and the active site is calculated to be 2e A assuming that the fluorescence of only one tryptophan per catalytic polypeptide chain is quenched by the acceptor or 27 A assuming that both tryptophans on a catalytic chain are equally quenched. The pyridoxamine phosphate label is also used as the fluorescent donor with mercurinitrophenol bound to the sulfhydryl group of the catalytic subunit as the energy acceptor. For this pair of labels the active site is determined to be very close to the sulfhydryl group on the same catalytic chain and 26 A from the sulfhydryl groups on the other chains of the catalytic trimer. In experiments with pyridoxamine phosphate at the active site as the donor and 8-anilino-1-naphthalenesulfonate at the active site as the acceptor, a distance of 26 A between active sites of a catalytic trimer is found. No energy transfer is observed from pyridoxamine phosphate at the active site to a fluorescamine derivative of cytidine 5'-triphosphate at the regulatory site. This implies that these groups are separated by at least 42 A in the native enzyme. All of the distances are calculated using the assumption of rapid rotation of donor and acceptor dipole moments relative to the donor fluorescence lifetime. Fluorescence polarization measurements suggest this assumption does not produce a significant error in the calculated distances. The distances between the various sites are related to the subunit structure of aspartate transcarbamylase.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase

Matsumoto¹,

Hammes²

1975

Biochemistry

View full text Add to dashboard Cite

show abstract

The Metallobiochemistry of Zinc Enzymes

Vallée

Galdes

1984

Advances in Enzymology - And Related Areas of Molecular Biology

175

View full text Add to dashboard Cite

The honour and opportunity of being invited to address you on the occasion of Professor Houtman's retirement is very much appreciated. However, having met him yesterday, I am convinced that I am not here to help celebrate his retirement as much as to initiate his new career.Just like careers, the role of metals in biology can be viewed from multiple points of view. In the past, metals have variously been thought to be beneficial, toxic or to serve no purpose at all in biology. It is not very long ago that documentation for the validity of all th ree perceptions seemed to coexist, and that they had equally vigorous advocates; but by now the hypothesis that they serve no biological purpose has faded into oblivion. This dilemma owed much to the fact that many metals are present in 'trace' amounts. I want to address myself today to the general principles underlying the evolution of metallobiochemistry and of its subdisciplines which did not exist a generation ago and give perspective to metals as toxic, environmental agents, a subject which has been of special interest to Prof. Houtman.The word 'trace' implies something that can be measured qualitatively but not quantitatively, and until about a generation ago that was true for many metals in biology. Therefore it was a nearly mystical subject explored of ten by individuals who had ideas about the problem but who performed few experiments to verify or reject them. This has changed. Measurement of very low concentrations of metals in biology has become quite routine. Technical problems have been eliminated to the point where the term 'trace' has become meaningless. That is fortunate, indeed, because to many 'trace' also meant 'unimportant' or 'insignificant' .Attention to the detection limits of analytical methods for metals has become almost irrelevant now. Yet, the fact that the II-B metals and those of the first transition series all occur in biological matter in similarly low concentrations led to the tacit inference that all of them would be found to perform similar functions. Such an emphasis on similarities of concentrations completely ignored the chemical properties of metals which are unique for each. Not too surprisingly, their biological roles have turned out to be as characteristic as their chemistries, enhanced by co-ordination of metals with 132 12 (1988) DELFT PROGRESS REPORT

show abstract

Histidin im aktiven Zentrum von Enzymen

Schneider¹

1978

Angew. Chem.

View full text Add to dashboard Cite

Aspartate Transcarbamylase from Escherichia coli

Cited by 86 publications

References 29 publications

Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase

Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase

The Metallobiochemistry of Zinc Enzymes

Histidin im aktiven Zentrum von Enzymen

Contact Info

Product

Resources

About