The Physical State Dependence of Carboxypeptidase A
            <sub>α</sub>
            and A
            <sub>γ</sub>
            Kinetics

Spilburg, Curtis A.; Bethune, J. L.; Vallée, Bert L.

doi:10.1073/pnas.71.10.3922

Cited by 28 publications

(25 citation statements)

References 19 publications

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“…It follows that crystals, constituted of conformations "selected" or "frozen" in this manner and static with respect to the free equilibration manifest in solution, could be expected to exhibit greatly reduced activities and markedly altered catalytic behavior, which indeed has been demonstrated for native carboxypeptidase a and y crystals (11). Clearly, the correlation of structure and function leading to enzyme mechanisms requires identity of the systems from which the respective data are derived.…”

Section: Discussionmentioning

confidence: 99%

“…Based on the spectral properties of the azoenzyme (4-6) and the nitroenzyme (15) and on the kinetics of the crystalline enzyme (11), we pointed out previously that the conformation of carboxypeptidase in solution differs significantly from that in crystals. In the correlation of structural crystallographic detail with the functional properties of the enzyme, multiple inconsistencies have come to light, particularly when trying to account for the properties of various, chemically modified carboxypeptidases in solution (7).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of multiple conformational states in solution.

Harrison¹,

Auld²,

Vallée³

1975

Proc. Natl. Acad. Sci. U.S.A.

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The red azoTyr-248.Zn complex of arsanilazocarboxypeptidase, previously used to demonstrate differences in conformation of the enzyme in crystals and in solution, has now provided means to detect multiple conformations of the enzyme in solution by stopped-flow pH and temperature jump experiments. These studies identify two distinct processes. Er + H+ ;=t Ey (I) (4, 7). The red azoTyr-248-Zn complex of azocarboxypeptidase now has proven to be a means of inspecting catalytic events and detecting multiple conformations in solution by stopped-flow, pH jump, and temperature jump experiments. Under one set of conditions, the rapid disruption of the complex due to substrate binding can be visualized directly by the disappearance of the red color (8, 9). Under another set of conditions, the probe identifies a pH independent equilibrium between two carboxypeptidase conformers, neither of which is red. These findings serve as a general model that may account for quantitative differences of conformations in solution, in crystals, and in different crystal habits. MATERIALS AND METHODSCarboxypeptidase Aa (Sigma Chemical Corp.) and Ay (Worthington Biochemical Corp.) were modified with diazotized arsanilic acid according to published procedures (4, 6, 7). Both enzyme forms gave analogous results. All other chemicals were reagent grade. Precautions to prevent contamination by adventitious metal ions (10) were taken throughout. Stock solutions of azoenzyme, 5 X 10-4 M, were prepared in 1 M NaCl, pH 7, and after centrifugation, diluted into appropriate, degassed solutions prior to stopped-flow experiments.Stopped-flow experiments were performed with a Durrum-Gibson instrument equipped with a Durrum fluorescence accessory no. 16400, a 75-W Xenon lamp and an endon EMI 9526B photomultiplier. The instrument was calibrated with a Cary 14 recording spectrophotometer to yield analogous spectra under rapid kinetic conditions. identical data were obtained with a Durrum stopped-flow instrument equipped to read absorbance directly, kindly furnished by

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of multiple conformational states in solution.

Harrison¹,

Auld²,

Vallée³

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…However, with the development of methods for cross-linking crystals, the insoluble crystalline preparations thus formed were studied in dilute buffer solution under conditions used for soluble enzymes (11,12,16). The majority of the studies involving cross-linked enzyme crystals have been restricted to hydrolytic enzymes and to the best of our knowledge, the preparation of cross-linked dehydrogenase crystals has not been reported previously.…”

Section: Introductionmentioning

confidence: 99%

Enzymatically active, cross-linked pig heart lactate dehydrogenase crystals

Bayne

Ottesen

1976

Carlsberg Res. Commun.

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Pig heart lactate dehydrogenase crystals were cross-linked with octanediimidic acid dimethylester. The resulting cross-linked crystals were insoluble in dilute phosphate buffer and had retained a high specific activity. Amino acid analyses showed that about twelve lysine residues out of twenty-three per subunit had been chemically modified. The cross-linked crystals showed increased stability in urea compared to the soluble enzyme. The differences caused by the cross-linking process to the properties of the enzyme are briefly discussed.

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“…With one exception, all functional residues which have been implicated as components of the active site of carboxypeptidase A (32,33) are preserved in carboxypeptidase B. These include in particular the three Zn ligands, His-69, His-196, and Glu-72 (3), Glu-270, Tyr-248, Arg-145, and Tyr-198 (32,33 …”

Section: Discussionmentioning

confidence: 99%

“…These include in particular the three Zn ligands, His-69, His-196, and Glu-72 (3), Glu-270, Tyr-248, Arg-145, and Tyr-198 (32,33 …”

Section: Discussionmentioning

confidence: 99%