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Prephenate dehydratase has been purified 10,000-fold from the crude extracts of Bacillus subtilis. The procedure takes advantage of the dissociation of the enzyme to a 55,000-dalton form in the presence of the negative effector, phenylalanine, and its association to a 210,000-dalton form in the presence of the positive effector, methionine. These two forms of the enzyme were separated from the bulk of the other proteins present in the crude extracts by gel filtration alternately in the presence of the two effectors. Sodium dodecyl sulfate electrophoretic analysis showed the enzyme is composed of apparently identical 28,000-dalton polypeptides.

A series of potential affinity adsorbents for prephenate dehydratase were synthesized by coupling allosteric effectors to agarose with and without spacer arms. Only cationic ligands containing a hexamethylene spacer arm adsorbed the enzyme. Activity could be removed from aminohexyl- and glycyl-aminohexyl-agaroses with 0.5 M NaCl (with loss of activity), 0.4 M phosphate, or 140 mM hexanediamine, but was not removed from ala-aminohexyl-, phe-aminohexyl-, met-aminohexyl-, leu-aminohexyl-, or phenylpyruvyl-aminohexyl-agaroses or by Gly, Ala, Phe (inhibitor), Met or Leu (both activators). Urea (1.0 M), 1 mM phosphate, 50% glycerol in phosphate or prephenic acid (substrate) did not release enzymatic activity from any of the gels. The gel-bound enzyme was found to have enhanced activity comparable to that of the methionine-activated soluble enzyme. The site and mode of the enzyme immobilization are discussed in relation to the possible hydrophobic/ionic subunit-subunit interactions.

Three active transport systems for amino acids and another for glucose in germinated conidia of Neurospora were all inactivated during incubation with the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetamide. Where tested, the inactivation by p-chloromercuribenzoate was reversed by the action of dithiothreitol. However, two other sulfhydryl reagents, the chloromethyl ketone derivatives of leucine and lysine, were found to have differential effects on the various transport systems. Inactivation by these two compounds did not occur at the active site of the permeases by affinity labeling, suggesting differential availability of sulfhydryl groups among the transport systems. Also, inactivation by these reagents was greatly enhanced in the presence of p-chloromercuribenzoate.

A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.

A number of methyl ketones have been prepared from l -leucine and found to be competitive inhibitors of Aeromonas aminopeptidase. These inhibitors were leucine methyl ketone (K i 18 μ m ), leucine chloromethyl ketone (K i 0.67 μ m ), and leucine bromomethyl ketone (K i 0.20 μ m ), and the corresponding succinimido derivative (K i 170 μ m ), succinamic acid derivative (K i 6.9 μ m ) and phthalimido derivative (K i 140 μ m ). Reversible inhibition was observed for all of the inhibitors tested, indicating that the active site of this enzyme is not alkylated or acylated by the nucleophile-sensitive components of some of the inhibitors. The chloromethyl ketones derived from l -leucine and l -phenylalanine were found to have the same relative binding constants as the substrates, l -leucinamide and l -phenylalaninamide.

The photochemistry of phenacyl, 1-, and 2-naphthacyl α-chymotrypsins has been studied using light of wavelengths greater than 310 nm. Irradiation of these modified enzymes, having aracyl chromophores covalently bound at Met-192, led to partial regeneration of their esterase activity. The initial rates of photoreactivation have been found to be dependent on the hydrogen ion concentration and on the presence of potential competitive inhibitors having triplet energies lower than the chromophores initially receiving the light energy. Irradiation of carbonyl-14C-labeled phenacyl α-chymotrypsin resulted in partial loss of this radiolabel from the protein at a rate concurrent with the increase in esterase activity. These results have been interpreted and discussed in terms of a dual photochemical behavior of these modified enzymes involving (1) cleavage of the Met-192 sulfur-phenacyl α-carbon bond leading to liberation of α-chymotrypsin and substituted phenone(s) and (2) photoafiinity labeling of one or more of the functional groups in the enzyme active-site region.

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.

THE similarity of major biochemical pathways throughout the animal and plant kingdoms1 presumably reflects the ancient formation of the pathways in a common ancestral counterpart of modern cell types. The pathway previously known for L-tyrosine biosynthesis in both prokaryotie and eukaryotic microorganisms (Fig. 1a) involves a dehydrogenation-decarboxylation conversion of prephenate to 4-hydroxyphenylpyruvate followed by transamination in the presence of a suitable amino donor. Our data (Fig. 1b) demonstrate a reversed order of these two reactions in several species of blue–green algae (more appropriately designated bacteria (order Chrooeocales2)).

Kinetic analyses of the irreversible inhibition of l -tyrosine and l -phenylalanine transport in Bacillus subtilis by phenylalanine chloromethyl ketone revealed that the inhibition was due to an affinity labeling process. Phenylalanine chloromethyl ketone is a competetive inhibitor of l -tyrosine and l -phenylalanine transport. The K i values for irreversible inhibition of l -tyrosine and l -phenylalanine transport were 194 and 177 μ m , respectively, and the first order rate constants for the alkylation reaction leading to inactivation of transport of l -tyrosine and l -phenylalanine were 0.016 and 0.012 min −1 , respectively. The similarity of these constants are consistent with the involvement of the same functional site for l -phenylalanine and l -tyrosine transport. A second effect of phenylalanine chloromethyl ketone was inhibition of the uptake of neutral, aliphatic amino acids; transport of basic and acidic amino acids was unaffected by it. Since high concentrations of any amino acid did not reduce the inhibitory effects of phenylalanine chloromethyl ketone on transport of neutral, aliphatic amino acids, an independent effect, not due to an affinity labeling process was inferred. A procedure for selective labeling of the l -tyrosine/ l -phenylalanine transport system was demonstrated that should be applicable to the introduction of a radioactive label into the transport protein(s).

Coupling the cathepsin D inhibitor pepstatin to poly( l -Lys) ( M r 13,000) is shown to enhance its inhibition of protein breakdown in whole cell systems. Rates of intracellular protein breakdown for prelabeled proteins of Balb/c 3T3 fibroblasts were measured in the presence and absence of amino acids and insulin to generate basal and enhanced rates of protein breakdown. Pepstatin and poly( l -Lys) inhibited rates of degradation 5–7% and 16–23%, respectively, under each condition. Pepstatinyl-poly( l -Lys), containing 9 mol pepstatin/mol polymer, inhibited enhanced rates of degradation a further 24–33% compared to poly( l -Lys), but this extra increment was not seen under basal conditions. Although the mechanism of inhibition of intracellular protein breakdown by poly( l -Lys) presently is unknown, the data obtained with free and conjugated pepstatin indicate the lysosomal system degrades proteins under both basal and enhanced conditions.

l -Tyrosine and l -phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l -tyrosine or l -phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l -tyrosine or l -phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l -phenylalanine and l -tyrosine is strictly competitive. Neither l -tyrosine nor l -phenylalanine uptake was competitively inhibited by other naturally occurring l -amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l -tyrosine and l -phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l -tyrosine and l -phenylalanine to inhibit the uptake of l -[ 14 C]tyrosine and l -[ 14 C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the α-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged α-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l -tryptophan and l -histidine, do not compete with l -tyrosine and l -phenylalanine for uptake. The presence of the p -hydroxy substitutent in the side chain (as in l -tyrosine) enhances the uptake of the aryl amino acid analogues investigated.

A transport double mutant of Neurospora crassa has been isolated that has only one of the three transport systems capable of l -histidine uptake. The substrate specificity of the remaining transport system, termed the general transport system, has been fully characterized with regard to the contributions to binding of the side chain, the α-amino group, and the carboxylate group. The positively charged α-amino group is necessary for binding; the negatively charged carboxylate group is of less importance, since its replacement by a neutral carbonyl functional group does not completely abolish binding. The greatest structural latitude for binding was found in the side chain; affinity for α-amino acids was uniformly high except for l -aspartic and l -glutamic acids, l -asparagine, and l -proline. Thus, this transport system is “general” with these restrictions.