Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.
A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.
Eisenstein
E
E
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Han
M S
MS
Woo
T S
TS
Ritchey
J M
JM
Gibbons
I
I
Yang
Y R
YR
Schachman
H K
HK
D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D001221:Aspartate Carbamoyltransferase