Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: A medium-resolution hydrogen exchange study

Journal Article · Tue Jun 27 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00439a011· OSTI ID:5364284

Mallikarachchi, D; Burz, D S; Allewell, N M ^[1]

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1. Wesleyan Univ., Middletown, CT (USA)

Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards and Englander et al., exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.

• OSTI ID: 5364284
• Journal Information: Biochemistry; (USA), Vol. 28:13; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
NUCLEOTIDES
BIOCHEMICAL REACTION KINETICS
PEPTIDES
MOLECULAR STRUCTURE
AMINO ACID SEQUENCE
ATP
CYTOSINE
ESCHERICHIA COLI
ISOTOPE DILUTION
LIQUID COLUMN CHROMATOGRAPHY
SOLUTIONS
TRANSFERASES
TRITIUM COMPOUNDS
AMINES
AZINES
BACTERIA
CHROMATOGRAPHY
DISPERSIONS
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
MICROORGANISMS
MIXTURES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
PROTEINS
PYRIMIDINES
REACTION KINETICS
SEPARATION PROCESSES
TRACER TECHNIQUES
550201* - Biochemistry- Tracer Techniques