Relaxation spectra of aspartate transcarbamylase. Interaction of the native enzyme with cytidine 5'-triphosphate

Previous evidence, from both crystallographic and biochemical studies, has indicated that profound tertiary and quaternary changes in the structure of Escherichia coli aspartate transcarbamylase occur upon the binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA). In particular, within a single catalytic polypeptide chain, the aspartate binding domain relocates closer to the carbamyl phosphate binding domain, thereby resulting in a major reorganization of the interface between the two domains. Among the new interactions, salt bridges between Glu-50 and both Arg-167 and Arg-234 are formed. In the present study, site-directed mutagenesis is used to replace Glu-50 by glutamine in the catalytic chain. The Michaelis constant for aspartate of the mutant catalytic subunit is about 10-fold higher and the turnover number 10-fold lower than their respective counterparts in the wild-type catalytic subunit, whereas the dissociation constant for carbamyl phosphate is almost unchanged. For the holoenzyme, this substitution results in an 8-fold decrease in the specific activity, a 20-fold increase in the aspartate concentration that gives half of the maximal velocity, and a loss of cooperativity for both substrates. However, the mutant enzyme is not "frozen" in a low-affinity-low-activity conformation since PALA stimulates the activity severalfold and induces an increase in the sulfhydryl reactivity analogous to that of the wild-type enzyme. Together these results indicate that the stabilization of the aspartate binding domain near the carbamyl phosphate binding domain, through specific interdomain bridging interactions, is necessary for the high-affinity-high-activity configuration of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Discussionsupporting

confidence: 82%

Relationship between domain closure and binding, catalysis, and regulation in Escherichia coli aspartate transcarbamylase

Ladjimi¹,

Middleton²,

Kelleher³

et al. 1988

“…Although the results reported here cannot be directly compared with those from earlier work, they are qualitatively similar. The simplest mechanism found in previous studies (Harrison and Hammes, 1973;Wu and Hammes, 1973) to be consistent with the kinetics of the binding of ligands to the regulatory site is shown in eq 2. In this mechanism Ej represents the "tight" Et 4-I el + i *£…”

Section: Results and Treatment Of Datamentioning

confidence: 74%

“…All solutions contained 2 mM carbamyl phosphate, 10 mM succinate, 0.15 M KC1, and 0.02 M imidazole-HCl (pH 7) in a final volume of 2 ml. The relaxation times and amplitudes were calculated using a PDP-11 digital computer on-line to the temperature-jump apparatus (Hilborn et al, 1973;Harrison and Hammes, 1973). The relaxation times reported are averages of five or six experiments.…”

Section: Methodsmentioning

confidence: 99%

Interaction of aspartate transcarbamylase with 5-bromocytidine 5'-tri-, di-, and monophosphates

Tondre¹,

Hammes²

1974

Self Cite

The interaction of the 5-bromo derivatives of CTP, CDP, and C M P with aspartate transcarbamylase from Escherichia coli was studied by binding, inhibition, and temperature-jump kinetic experiments in the presence of saturating concentrations of carbamyl phosphate and succinate, an aspartate analog. Almost the entire range of fractional saturation of regulatory sites could be studied with these three inhibitors. All three inhibitors display apparent negative cooperativity in their binding isotherms. The binding isotherms can be quantitatively described by a model consisting of two sets of three identical sites per enzyme or by a mathematically equivalent model of six identical sites per enzyme molecule with occupation of the first three sites weakening ligand binding to the T h e enzyme aspartate transcarbamylase from Escherichia coli is an allosteric enzyme inhibited by CTP and activated by ATP (Gerhart and Pardee, 1962). It contains distinct regulatory and catalytic subunits (Gerhart and Schachman, 1965). The native enzyme consists of six catalytic and six regulatory subunits (Weber, 1968; Hammes et ai., 1970;Meighen et al., 1970;Winlund and Chamberlin, 1970;Rosenbusch and Weber, 1971;Matsumoto and Hammes, 1973;Gray et al., 1973). Equilibrium binding studies have shown that CTP and ATP bind to the regulatory sites heterogeneously (Winlund and Chamberlin, 1970; Buckman, 1970;Matsumoto and Hammes, 1973;Gray et al., 1973). The data are consistent with a model postulating the existence of two classes of regulatory binding sites, three having a high affinity for the nucleotide effectors and three having a relatively low affinity. A second, equivalent, model is the postulation of six identical sites with the binding of the first effector molecule inducing negative cooperativity in the binding of a second molecule to a regulatory dimer. The amount of inhibition and activation appears to be directly proportional to the number of regulatory sites occupied (Matsumoto and Hammes, 1973).Kinetic investigations of the interaction of aspartate transcarbamylase with 5-bromocytidine 5'-triphosphate (Eckfeldt et al., 1970), CTP (Harrison and Hammes, 1973), the ATP analog 6-mercapto-9-P-D-ribofuranosylpurine 5'-triphosphate (Wu and Hammes, 1973), carbamyl phosphate (Hammes and Wu, 1971a), and succinate and malate (Hammes and Wu, 1971b) have been carried out. The results obtained suggest at least three conformational changes are involved in the control process. The binding of effector molecules can be explained by a mechanism assuming two classes of binding sites. However, in the cases of 5-bromo-CTP and CTP, kinetic experiments last three sites (i.e., negative cooperativity). The maximum inhibition and binding affinity of the ligands vary in the order 5-bromo-CTP > 5-bromo-CDP > 5-bromo-CMP. The results obtained from binding and inhibition experiments show that the fractional extent of inhibition is directly proportional to the fraction of regulatory sites occupied. A single relaxation process is observed for the enzyme-inhibitor...

“…In previous studies (Gray et al, 1973;Harrison & Hammes, 1973;London & Schmidt, 1974), the origin of the binding heterogeneity has not been definitively determined although ligand-induced asymmetry (negative cooperativity) has generally been the favored explanation. Our data suggest that regardless of the origin of the inequivalence any differences in conformation or any asymmetry in chain arrangement, whether preexisting or ligand induced, must be relatively small and local in nature.…”

Section: Discussionmentioning

confidence: 99%

Binding of regulatory nucleotides to aspartate transcarbamylase: nuclear magnetic resonance studies of selectively enriched carbon-13 regulatory subunit

Moore¹,

Browne²

1980

Specifically enriched [gamma-13C]phenylalanine, -tyrosine, and -histidine have been biosynthetically incorporated into aspartate transcarbamylase from Escherichia coli. These nonperturbing NMR probes have been used to characterize the interaction of the regulatory sites on the enzyme with nucleotide effectors. The C gamma carbons of the three tyrosines and four histidines per regulatory chain give narrow, well-resolved resonances, and the signals from the five phenylalanines per chain are partially resolved in the presence of bound inhibitor. Spectral changes in regulatory subunit were monitored as a function of concentration of the inhibitor, CTP, and the activator, ATP. Three histidine residues responded to ATP and CTP in an identical manner while two phenylalanine residues were sensitive to CTP but not ATP binding. The tyrosine resonances were not perturbed by effectors. The chemical shift response of the single observable histidine resonance to bound nucleotides in the reconstituted enzyme was identical with that observed for isolated regulatory subunit. This histidine spectrum was undisturbed by the T to R conformational transition of the enzyme. The results suggest that the regulatory subunit experiences minimal rearrangement of tertiary structure on binding effectors and that at least one phenylalanine and one histidine residue are present in the region of the CTP binding site.