The Catalytic Activity of Carboxypeptidase-degraded Aldolase

Drechsler, Edward R.; Boyer, Paul D.; Kowalsky, Arthur

doi:10.1016/s0021-9258(18)69749-2

Cited by 190 publications

(32 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The value, -23°, for [a]D is in agreement with that obtained by others (Jirgensons, 1959;Drechsler et al, 1959) for native aldolase and is in the range observed for many compact, globular proteins (Schellman gives the protein concentration directly in fringes (40 fringes = 10 mg/ml). A, native aldolase at 5 mg/ml in 0.05 m phosphate buffer pH 7.0.…”

Section: Resultssupporting

confidence: 90%

“…Difference spectra for both the acid and urea-dissociated aldolase compared to native aldolase as a reference solution are shown in Figure 5A. A similar acid difference spectrum has been reported previously (Drechsler et al, 1959). In these difference spectra maxima occurred at 279 and 287 mp, which are normally associated with tyrosyl residues; at 291 mp, attributable generally to tryptophanyl residues; and at 265 and 269 mp, corresponding to phenylalanyl residues.…”

Section: Resultssupporting

confidence: 79%

See 1 more Smart Citation

The Dissociation and Reconstitution of Aldolase^*

Stellwagen¹,

Schachman²

1962

Biochemistry

208

View full text Add to dashboard Cite

Rabbit muscle aldolase, which exists in solution as essentially globular, compact, highly organized particles of molecular weight 1.42 X 106, was readily dissociated into three polypeptide chains apparently without the rupture of covalent bonds. This dissociation was effected by treatment with any of the reagents, urea, HC1, acetic acid, or sodium dodecyl sulfate. Detailed studies of the subunits generated in 4 m urea solutions at pH 5.5 and in acid solutions (pH 2) showed that the polypeptide chains were markedly disorganized as coil-like particles of weight-average molecular weight 0.46 X 105. Upon dissociation and denaturation the reduced viscosity increased from 4.0 ml/g for the native enzyme to 23 ml/g at pH 2 and 18 ml/g in 4 m urea solutions. The optical rotation, [a]d, changed from -23°for the native protein to -62°and -83°for the aciddissociated and urea-dissociated subunits respectively. Similarly there was a marked change in the Drude constant, Ac, from 283 mp to 238 mp and 223 m^i. Accompanying the change in the conformation of the polypeptide chains was a blue shift in the absorption spectrum with AmaI decreasing from 279.7 mp for the native protein to 277.0 mp for the subunits. Analyses of the shift by difference spectra revealed contributions from chromophoric groups of tyrosine, tryptophan, and phenylalanine due to the altered environment of these chromophores in the dissociated chains as compared to the intact enzyme. Titration of the sulfhydryl groups showed that the 16 groups which are "masked" in the native enzyme reacted readily in the dissociated subunits.No enzymic activity could be detected in the subunits in the presence of urea. However, activity was regained with yields of 65% when the dissociating agents (hydrogen ions or urea) were removed by dilution or dialysis. Reconstitution experiments were performed under different conditions; pH 5.5 and concentrations greater than about 50 /il/ml were found to be optimal for the restoration of activity. Kinetic studies showed that extensive annealing was unnecessary, for enzymic activity was regained in only a few minutes. The reconstituted protein was characterized by its reduced viscosity, sedimentation coefficient, molecular weight, optical rotatory dispersion, absorption spectrum, titratable sulfhydryl groups, and specific activity. In all respects the reconstituted macromolecules were virtually identical to the native enzyme.

show abstract

Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 79%

The Dissociation and Reconstitution of Aldolase^*

Stellwagen¹,

Schachman²

1962

Biochemistry

208

View full text Add to dashboard Cite

show abstract

“…The enzymatic logic of the EMP and the EDP are broadly similar; glucose is taken up and phosphorylated, and, following a series of downstream transformations distinct to each pathway, the product is cleaved in a reverse aldol condensation reaction to yield two three-carbon compounds. However, the substrate of the aldol cleavage is different; in the case of the EMP, fructose 1,6- bis phosphate is cleaved to yield glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate, whereas in the EDP, 2- keto -3-deoxy-6-phosphogluconate (KDPG) is cleaved to yield G3P and pyruvate ( Kovachevich and Wood, 1955a , b ; Drechsler et al, 1959 ; Peekhaus and Conway, 1998 ). In both the EMP and the EDP, the metabolic fate of G3P following the aldol cleavage step is identical.…”

Section: Introductionmentioning

confidence: 99%

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

et al. 2021

View full text Add to dashboard Cite

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.

show abstract

“…Οι περιπτώσεις άμεσης μέτρησης ενός αντιδρώντος ή προϊόντος της ενζυμικής αντίδρασης για τον προσδιορισμό της ενεργότητας είναι σπάνιες. Για παράδειγμα, η ενεργότητα της β-γαλακτοσιδάσης μπορεί να προσδιοριστεί σύμφωνα με την παρακάτω αντίδραση [19]: [20] : αλδολάση 1,6-διφωσφορική φρουκτόζη--------------> φωσφορική διυδροξυακετόνη ( 1.20) μετριέται στα 240 nm.…”

Section: προσδιορισμός των σταθερών κμ και Vmaunclassified

Εφαρμογές Ακινητοποιημένων Ενζύμων Σε Οπτικές Και Ηλεκτροχημικές Μεθόδους Ανάλυσης

Καραλέμας¹

View full text Add to dashboard Cite

The Catalytic Activity of Carboxypeptidase-degraded Aldolase

Cited by 190 publications

References 31 publications

The Dissociation and Reconstitution of Aldolase^*

The Dissociation and Reconstitution of Aldolase^*

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Εφαρμογές Ακινητοποιημένων Ενζύμων Σε Οπτικές Και Ηλεκτροχημικές Μεθόδους Ανάλυσης

Contact Info

Product

Resources

About

The Catalytic Activity of Carboxypeptidase-degraded Aldolase

Cited by 190 publications

References 31 publications

The Dissociation and Reconstitution of Aldolase*

The Dissociation and Reconstitution of Aldolase*

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Εφαρμογές Ακινητοποιημένων Ενζύμων Σε Οπτικές Και Ηλεκτροχημικές Μεθόδους Ανάλυσης

Contact Info

Product

Resources

About

The Dissociation and Reconstitution of Aldolase^*

The Dissociation and Reconstitution of Aldolase^*