NDLI: Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Content Provider	Journal of Biological Chemistry (JBC)
Author	Macol, Christine P. Tsuruta, Hiro Kantrowitz, Evan R.
Description	The mutant catalytic subunit in which 6 aspartate residues have been added to the C terminus of each catalytic chain (AT-C) The reconstituted mutant holoenzyme in which 6 aspartate residues have been added to the C terminus of each catalytic chain ((AT-C)2R3) Mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Arg-105 in each catalytic chain and the other catalytic subunit has 6 aspartic acid residues added to the C terminus of each catalytic chain ((R105A-C)(AT-C)R3) Mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Arg-54 in each catalytic chain and the other catalytic subunit has 6 aspartic acid residues added to the C terminus of each catalytic chain ((R54A-C)(AT-C)R3)
Abstract	Two hybrid versions ofEscherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.
Related Links	http://www.jbc.org/content/277/30/26852.abstract
Ending Page	26857
Starting Page	26852
Page Count	6
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	30
Volume Number	277
DOI	10.1074/jbc.M203431200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2002-07-26
Access Restriction	Open
Subject Keyword	N-(phosphonoacetyl)-l-aspartate (PALA) The aspartate concentration at half-maximal observed specific activity ([Asp]0.5) Catalytic subunit of aspartate transcarbamoylase composed of three catalytic chains (C) Regulatory subunit of aspartate transcarbamoylase composed of two regulatory chains (R) ENZYME CATALYSIS AND REGULATION
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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