Linked Life Data

8576114

Source:http://linkedlifedata.com/resource/pubmed/id/8576114

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1996-3-11
pubmed:abstractText
Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M. P., Mallikarachchi, D., Matthew, J. B., and Allewell, N. M. (1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N. M. (1995) Proteins Struct. Funct. Genet., in press). To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different. In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit. Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Schachman, H. K. (1992) Biochemistry 31, 6562-6569). The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
271
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1285-94
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase.
pubmed:affiliation
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't