The effect of chemical modification by diethylpyrocarbonate on substrate and effector binding to phosphoenolpyruvate carboxylase from Crassula argentea. Evidence for a histidine residue in the malate binding site

Title:

The effect of chemical modification by diethylpyrocarbonate on substrate and effector binding to phosphoenolpyruvate carboxylase from Crassula argentea. Evidence for a histidine residue in the malate binding site

Creator:

Meyer, C. R., Rustin, P., and Wedding, R. T.

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:715ed98c-b4b6-419b-b4c4-ff563bd6717b
uuid:715ed98c-b4b6-419b-b4c4-ff563bd6717b
urnnbn:urn:nbn:cz:aba007-000fe0

Type:

model:internalpart and TEXT

Format:

print

Description:

Chemical modification of purifíed phosphoenolpyravate carboxylase (PEPC) from the crassulacean acid metabolism plant Crassula argentea Thunb. with the histidyl reagent diethylpyrocarbonate (DEPC) resulted in a transient increase in activity followed by a decrease of activity with time. This biphasic response was observed when the modifíed enzyme was assayed at both low (sub-K,^ and saturating substráte (phosphoenolpyruvate, PEP) concentrations. There was an approximate 25- fold difference in the apparent rate constants for the activation and inhibition phases. This is in contrast to what we háve observed under similar conditions for the C4 enzyme from Zea mays L. for which only inhibition of activity occurs. Spectral studies indicated that up to 7 of the potential 20 histidine residues per subunit were modifíed, at least 3 of which were necessary for activity. The biphasic response of the Crassula enzyme was dependent on the concentration of DEPC. Progressively less inactivation was observed when modifying the enzyme with lower concentrations of DEPC. Chemical modification of PEPC with 75 pM DEPC resulted in a form of the enzyme with a lower K^, and higher This was concomitant with the modification of 4 histidines per subímit. Changes in the response of the enzyme to allosteric effectors were also observed; with modification the enzyme was desensitized to malate inhibition and glucose-6-phosphate activation. The Kj of the modifíed enzyme for malate increased over 15-fold. This was consistent with fluorescence binding studies using the extrinsic conformationalprobe S-anilino-l-naphthalenesulfonate which indicated the elimination of binding of malate and increased binding of the substráte to PEPC. Protection studies showed that malate desensitization was delayed by the presence of malate during modification. Malate also slowed the initial rate of histidine modifícation as measured spectrophotometrically. Thus histidine plays a role in the malate site of Crassula PEPC.

Language:

Multiple languages

Rights:

http://creativecommons.org/licenses/by-nc-sa/4.0/
policy:public

Coverage:

259-272 and 101-114

Source:

Photosynthetica | 1993 Volume:28 | Number:2

Harvested from:

CDK

Metadata only:

false