Cofactor Requirements of Tryptophanase from Proteus rettgeri

Crystalline tryptophanase prepared from the cells of Proteus rettgeri is inactive in the absence of added pyridoxal phosphate. Half-maximal enzyme activity is obtained at a concentration of 1.81 µm. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 420 nm. Holotryptophanase requires K⁺ or for its maximal activity, but Na⁺ is inactive. No appreciable spectral change was observed on changing the ionic environments.

The amount of pyridoxal phosphate bound to the enzyme was determined by equilibrium dialysis and spectrophotometric titration to be 4 moles per mole of enzyme. Reduction of holoenzyme with sodium borohydride results in a shift of the absorption peak at 420 to 336 nm. ?-Pyridoxyllysine was isolated from the acid hydrolyzate of the reduced holoenzyme by paper chromatography and electrophoresis.

Addition of the substrate, l-tryptophan, or the competitive inhibitor, l-alanine, to the holoenzyme causes appearance of a new peak near 500 nm which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor. The similar spectral change was observed by the addition of pyruvate, ammonia and indole to the holoenzyme.     

l-amino acid oxidases of Proteus rettgeri.

Proteus rettgeri has been found to contain two separable 1-amino acid oxidases. Both enzymes are particulate in nature, neither being ribosomal bound. One of these enzymes appears to have broad specificity, being active toward monoaminomonocarboxylic, imino, aromatic, sulfur-containing, and beta-hydroxyamino acids. The other enzyme has more limited specificity, catalyzing the oxidative deamination of the basic amino acids and citrulline. The affinity of this oxidase for the various substrates at pH 7.6 in decreasing order is arginine, histidine, ornithine, citrulline, and lysine. This enzyme has a particularly high affinity for arginine (Km equal to 0.27 mM), and anomalous kinetics are observed with increasing substrate concentrations. When concentrations of arginine greater than 1.0mM were added to the reaction containing histidine, imidazole pyruvate formation was completely inhibited.     

Catalytic Properties of Tryptophanase from Proteus rettgeri

Formation of sclerotia in a strain of Sclerotinia libertiana Fuckel using Czapek-Dox agar medium was highest at pH 4.0~6.0 and at 22~25°C. The response was better under darkness than under light. However, when a potato-extract medium was used the optimum temperature range extended, and even at 15~27°C mature sclerotia formed. The addition of indole-3-acetic acid (IAA) to the Czapek-Dox medium containing riboflavine stimulated the formation of sclerotia under fluorescent light. Though iodoacetic acid, a ?SH reagent, also had a stimulatory effect, cysteine had little inhibitory effect on sclerotial formation. Formation was markedly inhibited by p-aminobenzoic acid (PABA), especially in the presence of tyrosine or tryptophan, and excess ammonium salts in the medium also produced an inhibitory effect. It was assumed that accumulation of an intermediary metabolite with high reactivity with ?SH groups was necessary for sclerotial formation, but PABA and excess ammonium salts inhibited the accumulation.     

Purification and some properties of beta-lactamases from Proteus rettgeri and Proteus inconstans

Two beta-lactamases were isolated from strains of Proteus species and purified, one from a strain of P. rettgeri and the other from a strain of P. inconstans. Each enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. Molecular weights of P. rettgeri and P. inconstans enzymes were found to be 42,000 and 43,000, and their isoelectric points pH 8.7 and 8.6, respectively. The two enzymes presented typical cephalosporinase profiles. Cefmetazole (CS-1170) and cefoxitin, both cephamycin antibiotics, not only resisted hydrolysis by both of the enzymes, but also inhibited their activities competitively. Rabbit antiserum against purified P. rettgeri enzyme inhibited the activity of both purified and crude enzyme preparations from other strains of P. rettgeri so far tested. None of the beta-lactamases produced by other species of Proteus including P. inconstans was inhibited by the antiserum, thus showing that the purified cephalosporinase was of the species-specific types. The enzymological properties of the preparations were compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Mechanisms of regulation of urease biosynthesis in Proteus rettgeri

Urease of Proteus rettgeri is an inducible enzyme synthesized specifically in the presence of urea; urea analogues did not act as inducers. Once initiated, the biosynthesis of the enzyme proceeded as a constant fraction of the total protein formed. The rate of urease formation was affected by the carbon source used. In comparison with glycerol, glucose inhibited enzyme synthesis. The addition of ammonium ions to the inducing medium also decreased the rate of urease biosynthesis, and when ammonium ions were present urease activity and urea transport across the cell membrane were inhibited. A kinetic analysis of urease inhibition by ammonium ions, by use of a partially purified preparation of urease, showed that it was a competitive inhibition.     

Regulation by repression of urease biosynthesis in Proteus rettgeri

Measuring the specific enzyme activity in cells of Proteus rettgeri it was shown that urease formation is controlled by repression through ammonia. Derepressed synthesis of the enzyme, as initiated by the absence of ammonia, required an external nitrogen source, which may not only be urea, but also nitrate, glutamate or nutrient broth. In contradiction to earlier reports the observations indicated that urea is not required for the synthesis of this enzyme, and that, therefore, urease is not an inducible enzyme in this microorganism.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Role of protein subunits in Proteus rettgeri penicillin G acylase.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.