The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)

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In all instances the reaction cis-aconitate-* citrate was studied. Citric acid was estimated by the method of Pucher, Sherman & Vickery (1936) as modified by Buffa & Peters (1949).

ACTION OF oc-AMYLASES 99 3. Nevertheless, under these conditions, some maltotriose, maltose and glucose are formed by fission of longer chains. The yield of glucose is far below that expected from a random fission. 4. The action of these enzymes on dilute amylopectin yielded, in the early stages, negligible traces of fission products of shorter chain length than 6-8 units. Salivary and fungal (A8pergillu8 oryzae) amylases under the same conditions yielded products of chain length of 3 units and upwards. 5. Dilute amylose in corresponding reactions yielded appreciable quantities of fission products of chain length of 2 units and upwards (malt and salivary) or 3 units and upwards (bacterial and fungal). 6. Salivary amylase does not readily attack the first two, and the bacterial and malt cx-amylases the first five linkages from the non-reducing end. 7. Salivary and malt oc-amylases readily attack the second and bacterial amylase the third linkage from the reducing end, other linkages nearer this end with increasing difficulty. It is concluded that linkages other than those near the ends here specified are attacked at random. 8. The action of the x-amylases on shorter linear chains, such as maltohexaose and maltotriose, is restricted as regards linkages near the non-reducing end so that fission must often take place nearer to the reducing end than is normal, and more glucose may be formed. 9. A hypothesis is suggested to explain the actions of the a-amylases. One of us (R. B.) is indebted to the Department of Scientific and Industrial Research for a grant, for which we express our thanks.

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Since the discovery of aeonitase by Martins and Knoop (1937), it has been generally accepted that citric acid is transformed into isocitric acid via cisaconitic acid. However. Martius and Lynen (1950) and Friedrich-Freska and Martius (1951) claimed that there was a direct conversion of citric aeid to wocitric acid, as they did not observe a latent period at the start of the reaction. Further, as a result of the mathematical treatment of the reaction velocities, Friedrich-Freska and Martius (1951) supposed that after combination of the substrate with the enzyme, there is formed an intermediate complex wliich can decompose into enzyme-cis-aconitic acid, enzyme-isocitric acid or enzyme-citrie acid. Tlicy regarded oi-s-aconitic acid as being only a by-product of the aeonitase reaction. These authors also studied the six initial reaction velociti® associated witli aconita.se activity and claimed that tlie difTcrence in the rates could account for the transient accumulation of isocitric acid above the equilibrium value that occurs when the reaction is started with cis-aconitic acid. On account of the agreement between their experimental findings and mathematical predictions, they concluded that a single enzyme could catalyse the conversion of eaeh of the three acids to the other two.The mathematical treatment which Friedrich-Freska and Martius (1951) applied to their results has been strongly criticised by Kacser (1952). Kreba and Ilolzach (1952) re-invesligated the problem of the lag period in the conversion of citric acid to isocitric acid, and showed that it did occur.As the studies on the kinetics of aeonitase had been made with crude or partially purified jireparations of the enzyme, the previous work was repeated with a more highly purified enzyme preparation (Morrison, 1954a). The question of the lag period was investigated, also the relative rates of the reactions catalyzed by aeonitase. The Michaelis constants of aeonitase for citric, cis-aconitic and isocitric acids were determined. For a preliminary communication see Morrison (1954b).

Jaeobsohn (1941) found tliat the pH optimum of aeonitase in the presence of phosphate buffer was in tlie neutral region. A more extensive study of the influence of pH on aeonitase aetivity was made by Johnson (1939). He elaimed that the pH-activity curve showed a sharp maximum at pH 7-4, but this value must be regarded as approximate. The pH values chosen for testing the aeonitase aetivity were widely spaced, and no mention was made of the buffers used to cover tlie extensive pll range.The above information was gained from studies with crude aeonitase preparations. Aconitase has now been highly purified (Morrison 1954a) and shown to be dependent on the presence of Fe-" and a redueing agent for maximum aetivity (Dickman and Clouticr, 1951;Morrison, 1954a). A more detailed investigation of tlip effect of pll on the activated, purified enzyme showed that the pH optimum is dependent on the type of buffer used. For a preliminary report of this work, see Morrison (iy54b).