The red azoTyr-248.Zn complex of arsanilazocarboxypeptidase, previously used to demonstrate differences in conformation of the enzyme in crystals and in solution, has now provided means to detect multiple conformations of the enzyme in solution by stopped-flow pH and temperature jump experiments. These studies identify two distinct processes. Er + H+ ;=t Ey (I) (4, 7). The red azoTyr-248-Zn complex of azocarboxypeptidase now has proven to be a means of inspecting catalytic events and detecting multiple conformations in solution by stopped-flow, pH jump, and temperature jump experiments. Under one set of conditions, the rapid disruption of the complex due to substrate binding can be visualized directly by the disappearance of the red color (8, 9). Under another set of conditions, the probe identifies a pH independent equilibrium between two carboxypeptidase conformers, neither of which is red. These findings serve as a general model that may account for quantitative differences of conformations in solution, in crystals, and in different crystal habits. MATERIALS AND METHODSCarboxypeptidase Aa (Sigma Chemical Corp.) and Ay (Worthington Biochemical Corp.) were modified with diazotized arsanilic acid according to published procedures (4, 6, 7). Both enzyme forms gave analogous results. All other chemicals were reagent grade. Precautions to prevent contamination by adventitious metal ions (10) were taken throughout. Stock solutions of azoenzyme, 5 X 10-4 M, were prepared in 1 M NaCl, pH 7, and after centrifugation, diluted into appropriate, degassed solutions prior to stopped-flow experiments.Stopped-flow experiments were performed with a Durrum-Gibson instrument equipped with a Durrum fluorescence accessory no. 16400, a 75-W Xenon lamp and an endon EMI 9526B photomultiplier. The instrument was calibrated with a Cary 14 recording spectrophotometer to yield analogous spectra under rapid kinetic conditions. identical data were obtained with a Durrum stopped-flow instrument equipped to read absorbance directly, kindly furnished by

The chromophoric intramolecular azoTyr-248.Zn complex detects discrete kinetic steps in the interaction of azocarboxypeptidase with products or substrates that are hydrolyzed slowly. Temperature-jump experiments at 510 nm indicate that the rapid binding of such ligands is followed by a slower change in the conformation of the enzyme--ligand complex: that defines the initial binding, and the rate constants k2 and k-2 for the forward and reverse steps of this conversion, respectively. For each ligand, the kinetically determined dissociation constant is virtually identical to that obtained at equilibrium form circular dichroic titrations. Although there are small variations in k2 and k-2 for each substrate, all the rate processes are much faster than the rate-determining step for the hydrolysis of these substrates. The proposed model of the mechanism of peptide hydrolysis by carboxypeptidase incorporates the results of these temperature jump experiments.

A kinetic study of the interaction between aspartate transcarbamylase and cytidine 5'-triphosphate in the presence and absence of carbamyl phosphate and succinate, an aspartate analog, has been carried out using the temperature-jump method. A single relaxation process was observed in the presence of carbamyl phosphate and succinate. The reciprocal relaxation time and amplitude of this process increase with increasing cytidine 5'-triphosphate concentration and reaches a limiting value at high concentrations. Two relaxation processes are observed in solutions containing enzyme and cytidine 5 '-triphosphate in the absence of carbamyl phosphate and succinate. The faster process apparently reflects the interaction of cytidine 5'-triphosphate with the catalytic sites while the slower process is due to binding at the regulatory sites. The reciprocal relaxation time of the slower T A he regulation of aspartate transcarbamylase from Escherichia coli by nucleotide effectors is a well-known example of allosteric control (Gerhart and Pardee, 1962). The enzyme is inhibited by CTP and activated by ATP, and is known to consist of regulatory and catalytic subunits (Gerhart and Schachman, 1965). Equilibrium binding studies have shown that CTP and ATP bind to the enzyme in a heterogeneous manner (Winlund and Chamberlin, 1970;Buckman, 1970;Matsumoto and Hammes, 1973). The data are consistent with the existence of two classes of binding sites, three having a high affinity for the nucleotide effectors and three having a relatively low affinity.Kinetic investigations of the interaction of aspartate transcarbamylase with 5-bromocytidine 5'-triphosphate (BrCTP;* 1 Eckfeldt et al., 1970), carbamyl phosphate (Hammes and Wu, 1971a), and succinate and L-malate (Hammes and Wu, 1971b) have been carried out. The results suggest that at least three conformational changes are involved in the control process. In this work, the results of an investigation of the interaction of CTP with aspartate transcarbamylase are presented. This study was undertaken because of the detailed equilibrium binding data now available (Matsumoto and Hammes, 1973). By using sensitive pH detection and by carefully choosing experimental conditions, a considerably wider concentration range could be examined than in the study using BrCTP (Eckfeldt et al., 1970). The results of this study show that a conformational change is rate limiting in the binding process in the presence of 1 mM carbamyl phosphate and 10 mM sucf From the

The intensely chromophoric intramolecular coordination complex formed between arsanilazotyrosine-248 and the active site zinc atom of azocarboxypeptidase A (Johansen, J. Proc. Nat. Acad. Sci. USA 68, 2532USA 68, -2535) is a spectrokinetic probe of catalytic events. The interconversion of the azoTyr-248.Zn complex and its constituents is measured by stopped-flow pH and temperature-jump methods. The rate of interconversion, 64,000 sect, is orders of magnitude faster than that of the catalytic step itself (about 0.01-100 sec'1). Rapidly turned over peptide and ester substrates disrupt the azoTyr-248.Zn comp ex before hydrolysis occurs. As a consequence, formation of azoTyr-248, substrate binding, and catalysis can all be monitored while catalysis is actually in progress. The results of these dynamic studies specify a course of catalytic events, different from those postulated based on x-ray structure analysis. If azoTyr-248 is displaced, the direction is opposite to the inward movement postulated on the basis of x-ray studies and is not unique to induction by substrates, since rapid changes in pH also result in analogous spectral changes.AzoTyr-248 carbox eptidase has all the features which are essential for mechanistic studies: (1) It is enzymatically active; (2) the spectra of the metal complex differ characteristically from those of its constituents; (3) it responds dynamically to environmental factors; and (4) the response time of the probe itself is much more rapid than is required for the measurement of the catalytic step. These combined kinetic and spectral properties of the metal complex render it a powerful spectrokinetic probe to visualize and discern microscopic details of the catalytic process. Spectral probes have proven very effective in exploring the relationship of catalytic activity to local structure of enzymes (1). They must be able to detect local changes in conformation, essential to and synchronous with catalysis, and must have highly specific characteristics with extraordinarily fast response times. We have employed site-specific inorganic and organic enzyme modifications to result in such sensors of electronic, magnetic, and structural changes accompanying catalysis (1). The intensely chromophoric intramolecular coordination complex between arsanilazotyrosine-248 and the zinc atom of azocarboxypeptidase (2, 3) has proven particularly informative in probing the vicinal perturbations, mutual orientation of, and distance between these two constituents of the active site of this enzyme. The spectra of this coordination complex respond dynamically to environmental factors, e.g., pH, substrates, inhibitors, and denaturing agents, as well as the physical state of the enzyme (4). Moreover, the spectra of the metal complex differ characteristically from those of its constituents and, further, the enzyme is fully active.In the present study temperature-jump and stopped-flow techniques have served to examine the rates of interaction of * To whom correspondence should be addressed. azoTyr-248...

Aspartate transcarbamylase from Escherichia coli has been prepared with up to four of zinc ions replaced by manganese, and the effect of this substitution on the proton nuclear magnetic resonance properties of succinate bound to the catalytic site and of cytidine 5'-triphosphate bound to the regulatory site has been determined, The specific activity and allosteric properties of the Mn-substituted enzyme are essentially identical with those of the native enzyme. The longitudinal relaxation time, T1, of the succinate protons is shortened by the native enzyme and is shortened further by the Mn-substituted enzyme at both 100 and 220 MHz in D2O solutions of 0.02 M immidazole chloride (pH 7.0), 10 minus 3 M beta-mercaptoethanol, 0;2 mM ethylenediamenetetraacetic acid, and 2.5 mM carbamyl phosphate over a temperature range of 5 to 35 degrees. Under the same conditions, the transverse relaxation time, T2, of the succinate protons at 90 MHz is shortened to the same extent by native and Mn-substituted enzyme. The temperature dependence of the relaxation times indicates that the shortening of the transverse relaxation time is determened by the lifetime of bound succinate, whereas the further shortening of the longitudinal relaxation time by the Mn-substituted enzyme is due to dipolar relaxation, i.e. to the interaction between Mn and the succinate protons. The distance between the Mn and the protons of succinate bound to the enzyme can be calculated from the relaxation time measurements and is 15,3 A. The dipolar interaction correlation time which is needed for the calculation of this distance, was found to be 3.5 X 10 minus 9 sec from the frequency dependence of T1. The transverse relaxation time of the C-6 proton of CTP is shortened to the same extent by both the native and Mn-substituted enzyme in D2O solutions of 0.02 M imidazole chloride (pH 7.0), 10 MINUS 3 M beta-mercaptoethanol, 0.2 mM ethylenediaminetetraacetic acid, and 2.5 mM carbamyl phosphate over the temperature 5-30 degrees. Since the temperature depencece of the relaxation time indicates the relaxation is not exchange limited, the manganese must be too distant from the bound CTP for an appreciable interaction to occur. This requires that the manganese be greater than 20A from the CTP. These results are used together with other available structural data to construct a schematic model for aspartate transcarbamylase.