Kinetics of substrate and product interactions with arsanilazotyrosine-248 carboxypeptidase A

Harrison, L. W.; Vallée, Bert L.

doi:10.1021/bi00614a001

Cited by 12 publications

(9 citation statements)

References 10 publications

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“…Arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]) (Johansen & Vallee, 1973Alter & Vallee, 1978;Harrison & Vallee, 1978) is a derivative with the selectivity modified chromophoric arsanilazotyrosine-248 residue introduced without significantly affecting the enzyme's catalytic properties. It has proven to be particularly useful for studying the conformations of the enzyme (Harrison et al, 1975;Hirose & Wilkins, 1984;Hirose et al, 1985a,b).…”

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confidence: 99%

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

Hirose¹,

Ando²,

Kidani³

1987

Biochemistry

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The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

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confidence: 99%

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

Hirose¹,

Ando²,

Kidani³

1987

Biochemistry

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“…The azo derivative has proven to be a sensitive, dynamic probe of environmental conditions (15,25,26,(32)(33)(34)(35)(36) and to respond to changes in pH and physical state of the enzyme with significant alterations in spectral properties. In the emulsion, the 510-nm absorption maximum is generated between pH 7.5 and 8.9 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The data Biochemistry: Thompson et al suggest that, at low temperatures, yet other physicochemical factors can determine aspects of enzyme structure that underlie spectral features of the azoenzyme. Thus, extensive conformational studies of azocarboxypeptidase (34)(35)(36), have revealed rapidly interconvertible substructures, involving both pHdependent (yellow red) and pH-independent (yellow yellow) processes (35,36). For instance, the decreasing temperature or increasing ice-like structure of supercooled water (10, 11) might conceivably force the enzyme into a predominantly "yellow" conformation, thereby causing the disappearance of the red color.…”

Section: Resultsmentioning

confidence: 99%

Structure and function of carboxypeptidase A alpha in supercooled water.

Thompson¹,

Gehring²,

Vallée³

1980

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

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The spectral and enzymatic characteristics of chromophoric derivatives of carboxypeptidase Aa (EC 3.4.17.1) have been examined at subzero temperatures in supercooled water-in-oil emulsions. Substrate and temperature dependencies of enzyme kinetics indicated the existence of a solution-like enzyme phase that greatly extends the temperature range (>60'C) over which the activity of this enzyme can be measured. The emulsion spectra were virtually identical to those of solutions over a wide range of temperatures. Subzero temperatures (<-10'C) may induce changes of enzyme conformation but not of geometry at the site of the metal atom, nor do they adversely affect activity at any of the temperatures studied. Both structure and function of carboxypeptidase Aa can be examined in supercooled water under identical reaction conditions. Theoretically, the ultimate verification of reaction mechanisms of enzymes demands the determination of their functional and structural characteristics with the same substrates under identical experimental conditions. In practice, however, the requisite experiments can be performed only with great difficulty, if at all. Generally, ideal circumstances for the performance of standard kinetic and thermodynamic experiments vary widely. Whereas rapid kinetics (1-3) call for excellent substrates, thermodynamic techniques are not only less demanding in this regard but, in fact, often utilize inhibitors or pseudosubstrates. Hence, the two approaches can result in quite divergent views of the formation, characteristics, and breakdown of a given enzyme-substrate complex. At present, the structural basis of enzymatic activity is generally deduced from time-averaged procedures at thermodynamic equilibrium, whereas functional conclusions often rest on pre-steady-state or steady-state conditions at "kinetic equilibrium." Owing to the vastly different time frames in which structure and function of enzymes can be assessed at conventional temperatures, it has proven most difficult to delineate the structure of the enzyme-substrate complex at the moment of catalysis.Reduction of temperature to subzero levels is an obvious potential means to reduce or eliminate this disparity. The establishment of conditions, media, and instrumentation for the study of enzymes in aqueous-organic cryosolvents (4-6) has led to the detection of transient species in enzyme reactions.However, organic solvents can adversely affect enzymatic reactions, of course, and both inhibition by solvent (7,8) and effects on substrate binding (9) are not uncommon. Hence, integration of data obtained at room temperature with those at subzero temperatures in organic solvents may generate problems.A water-in-oil emulsion (a reversed micellar system) provides a medium with a temperature range from at least +380C to -380C by supercooling water without freezing and without requiring changes in composition of that medium. Both the preparation of such emulsions and measurement of some of their physical properties have been described (10, 11)....

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“…A possible candidate may be tyrosine 248 in its ionized state as two conformations in solution have been observed for the azo-modified enzyme (Johansen & Valle, 1971). It has been shown that tyrosine 248 exists at least in two slowly interconverting states, of which one is close to the metal (Harrison &Vallee, 1978).…”

Section: (1 C) Aom Calculations Of Nqfsfor CD Carbonic Anhydrasementioning

confidence: 99%

Perturbed angular correlation spectroscopy and its application to metal sites in proteins: possibilities and limitations

Bauer¹

1985

Quart. Rev. Biophys.

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The present review describes the methodology of perturbed angular correlation of gamma rays (PAC) applied to biological problems. A large part of the present review focuses on the application of PAC spectroscopy to the study of coordination geometry for the metal site in zinc enzymes. Applications to conformation and dynamics of biomolecules and the use of the method to identify the intracellular sites of either metals or labelled proteins are also discussed.

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Kinetics of substrate and product interactions with arsanilazotyrosine-248 carboxypeptidase A

Cited by 12 publications

References 10 publications

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

Structure and function of carboxypeptidase A alpha in supercooled water.

Perturbed angular correlation spectroscopy and its application to metal sites in proteins: possibilities and limitations

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