Frederick C. Wedler

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Abstract Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l -Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [ 14 C]Asp C-Asp exchange is much faster than [ 32 P]carbamyl phosphate P i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E a (>15 °C) = 6.3 kcal/ mole and E a ( E a ≅ 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.

Two regulatory isozymes of glutamine synthetase from Bacillus caldolyticus, an extreme thermophile.

Abstract Two glutamine synthetases (EC 6.3.1.2) have been purified to homogeneity from B. caldolyticus strain YTP, grown at 70° on a minimal, defined medium. The enzymes are virtually identical in size and molecular weight (12 sub-units of MW 50,000), but differ in their isoelectric points, electrophoretic mobility, net charge, inherent thermal stability, affinity for substrates, and activity responses to metal ions and pH. Of primary interest is the observation that the more acidic form (pI = 5.2), EI, is strongly feedback-regulated by certain amino acids derived from glutamine (Gly, L-Ala, L- and D-ser) but not by L-Glu or AMP, whereas the less acidic form (pI = 5.5), EII, is inhibited most strongly by L-Gln and AMP, but not by the above amino acids. Both enzymes are inhibited strongly by ADP, CTP, NAD, glucosamine-6-P, less strongly by nucleotide diphosphates and L-Trp, and are activated by nucleotide monophosphates other than AMP. These results suggest that overall regulation of glutamine synthetase by the full spectrum of end product metabolites derived from L-Gln is accomplished by regulatory isozymes in this extremely thermophilic organism.

Modes of modifier action in E. coli aspartate transcarbamylase.

Abstract The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate Pi. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.

Mechanisms of enzyme control as probed by equilibrium exchange rates: Patterns of modifier effects with a two substrate, two product system

Abstract Measurements of reaction rates at equilibrium by isotopic exchange techniques can give considerable information about the mode of action of modifiers of enzymic reaction rates. To illustrate the various patterns that may be obtained, differing effects of modifiers on the exchange of A with P and of B with Q in the simple enzymic reaction of A + BP + Q are given. For this, reasonable values of rate constants are assumed, and calculations made for random and compulsory binding order systems. Cases where modifiers bind at the catalytic sites of substrate or at other binding sites, and where substrate association, substrate dissociation, covalent interconversion, or total catalytic capacity are modified are considered. Some quite distinctive patterns emerge, among the most interesting being those in which a modifier may block net catalysis yet allow one equilibrium exchange to occur essentially unhindered.

Spectroscopic probes of Escherichia coli glutamine synthetase. Rare earth ions by difference absorption.

Abstract 1. 1. Nd(III) and other tripositive lanthanide ions stimulate the biosynthetic activity of adenylylated Escherichia coli glutamine synthetase ( l -glutamate:ammonia ligase (ADP), EC 6.3.1.2) about 10–20% as well as Mn(II) at pH 6.50. With other forms of the enzyme and other assays, Nd(III) inhibits activity. 2. 2. Probe experiments with EPR and pulsed NMR, suggest that Nd(III) binds to the E 11 -(Mn 2+ ) 12 n1 enzyme at the n 2 sites to produce the E 11 -(Mn 2+ ) 12 1 −(Nd 3+ ) 12 n2 complex. 3. 3. Nd(III) bound to adenylated enzyme, compared to free ion, produces a difference absorption spectrum with relatively sharp peaks and troughs in the 300–600-nm region. 4. 4. This difference spectrum has been used to qualitatively evaluate the nature of the metal ion environment, and as a probe for studying perturbations by small molecules bound to the protein. The unique spectral changes induced by each substrate or modifier supports the conclusion that each possesses a unique binding site or mode of interaction with the protein.

Regulation of mammalian asparagine synthetase by adenine nucleotides.

Initial velocity kinetic data indicate that ADP and AMP are inhibitors of mammalian liver asparagine synthetase. The non-product nucleotide ADP is a much more potent inhibitor than AMP, although both apparently compete for the same site. This modifier site, however, does not overlap spatially with the substrate site for ATP. Both ADP and AMP are Vmax inhibitors, but ADP also raises the Km for ATP. Adenylate energy charge, calculated at various levels of ATP and ADP show typical correlations with activity, but with AMP these correlations are weak and atypical.

Evidence for cooperative Mn-ATP binding with Bacillus sp. glutamine synthetase.

Abstract The binding of Mn-ATP with B. subtilis glutamine synthetase, observed kinetically at 37°, pH 7.0, is cooperative (Hill n = 2.3, S 0·5 = 0.36mM) , a phenomenon overlooked in earlier studies. The Arrhenius plot is biphasic with a break at 26°C. Similar behavior is observed with the thermophilic B. stearothermophilus enzyme, but is absent with the enzymes from E. coli , plant, and mammaliam sources under optimal assay conditions. The temperature dependence of the intrinsic fluorescence of the protein is also non-linear, and the intersection point of 18° shifts to 30° upon binding of substrates. These results are interpreted as indicating that Bacillus sp . enzymes can assume multiple, functionally important conformational states related to Mn-ATP binding at 37°. They also emphasize further that critical differences in mechanism exist among glutamine synthetases from different sources.

Conformational states of chymotrypsin at high pH: substrate activation and sidechain interactions

A cross-over from specific to non-specific behavior occurs with amino acid substrate sidechain groups equal to or smaller than that of l-alanine, as revealed by the pH dependence of δ- and α-chymotrypsin-catalyzed hydrolysis of l-leucine and l-alanine derivatives. These data suggests that the −CH3 sidechain group, properly aligned, is large enough to sterically induce an active site conformation such that Kcat is pH independent above pH 7, in contrast to behavior observed with non-specific substrates, e.g. hippurates. Increased catalytic efficiency is apparently induced primarily by filling the remainder of the sidechain binding site with an appropriate aromatic moiety. The parameters kcat and Km were determined, plus the individual constants k2 and k3, separated by the technique of added nucleophiles. With the alanine derivative, substrate activation was observed, and is interpreted in terms of binding to a separate interacting non-catalytic site.

Continuous pH stat assay technique for glutamine and asparagine synthetase enzyme systems, involving ATP conversion to ADP + Pi and AMP + PPi, respectively

Abstract Glutamine synthetase and asparagine synthetase systems with reactions involving lysis of ATP to ADP and P i or AMP and PP i are usually assayed by discontinuous sampling and analysis or by coupled enzymic systems. Experimental results confirm theoretical predictions that such reactions may be continuously and directly monitored by pH stat devices. Sample volumes of 0.5–1.0 ml and buret volumes of 0.05–0.25 ml, with ATP levels near 1 m m can be used routinely. The number of enzyme reactions involving ATP to which this technique can be applied is quite large.