And to all the other members of the Kerckhoff Laboratories for Biology and of the Division of Chemistry whose assistance and kindness I enjoyed. Microbiologists - Kluyver not excluded (2) - have since turned their attention to another order of interpretation of variability, a more successful interpretation: the discrete, particulate one that results from the application of the principles of. Yet, while it seems to discreetly prevail in the highly organized world of the chromosomes (positional effects that are not permanent), its rule is fading in the realm of physiology and biochemistry.
With such thoughts in mind, we undertook the investigation of the metabolism of wild and mutated Neurospora strains in threonine province. The dry material was ground in a mortar and stored in a desiccator over calcium chloride in the cool box. essential differences in the enzymatic activities of the different batches prepared in the course of these studies.
INCUBATION AND ASS{1.Y METHOD~
The enzyme preparation is the supernatant after centrifugation of 30 mg lyophilisate per ml M/2 acetate-phosphate buffer, adjusted so that the pH of the reaction mixture would fall between 5.1 and 5.5. No inhibition of the deocarboxylation of alpha-ketobutyric acid by the addition of a 0.2 M NaBr solution was found, so there is no reason to believe that NaBr is present in the solution. The best separation of the 2,4-dinitrophenylhydrazones from alpha-tronbutyric acid, pyruvic acid, alpba-ketoglutaric acid and ketotbreonine was obtained using butanol saturated at 2%.
Effect of sample pH on Rfs of hydrazone Paper: Whatman #1. Where multiple values are given, this means that multiple spots appeared). We found no significant destruction of alpha-aminobutyric acid in the course of one hour of heating (Table IV), while the intensity of the fluorescence continued to increase during more prolonged heating. There is no general agreement on the nature of the prosthetic group of the deaminases involved.
EXTRACTION AND PURIFICATION
ALPHA-KETOBUTYRIC DECARBOXYLASE
Neuberg and Curb (65) found in 1912 tbat live yeast and lebedew juice were both capable of catalyzing the decarboxylation of alpha-ketobutyric acid as well as of pyruvate. Using washed dry yeast, Peters (66) obtained a 20-fold increase in alpba-ketobutyric decarboxylase activity by adding cocarboxylase. We have found that Neurospora extracts are able to decarboxylate alpha-ketobutyric acid at a 1-at similar to that of pyruvic acid decarboxylation.
The distillate from a reaction mixture in which alpha-ketobutyric acid has been decarboxylated was taken up in a 2,4-dinitrophenylhydrazine solution. A precipitate came down, which after filtering and washing could not be. distinguished from a known sample of 2,4-dinitrophenylhydrazone of propionaldehyde when chromatographed using the method we used for keto acid analysis. By dialyzing a Neurospora extract overnight against pH 8.2 phosphate buffer, a cocarboxylase effect could be detected (Fig. 13).
The alpha-ketobutyric decarboxylase activity of the preparation dropped to about one-fourth of its original value after it was switched and cocarboxylase and .manganese ions were added. When we tried to remove all the cocarboxylase from the preparation by dialyzing for longer time and/or at higher pH*, we lost all the decarboxylase activity and did not regain it by adding cocarboxylase and manganese ions. We used the same dialyzed preparation that activated cocarboxylase addition, to determine the effect of pH on the rate of decarboxylation (fig. 14).
Such a decrease in decarboxylase activity from pH 5.5 to pH 5.1 as found in this experiment was not encountered in other experiments in which undialyzed preparations were used. In connection with our efforts to elucidate why the in vivo metabolism of alpha-ketobutyrlc acid differs in 44104 from that in other strains, we tested the eight extracts of cultures of the wild type and 44104 mentioned. WOZ'e tii.oo zero ( tiill8 when the substrate wo.s added) gas exchange tms practical.J,y n.U in el.1 vsssels.
This teet was performed in the same way as with the same. ingx>edisnts. of the enzyme preparation is titrated to the desired pH. VI) DISCUSSION A) RE IN VITRO METABOLISM.
Extracts of the pyridoxine-less strain 44602, while deaminating fu.reonine at a subnormal rate, were not activated by pyridoxal phosphate addition (table XVIII). Addition of alpha-ketoglutaric acid did not affect the course of threonine dearnination. The course of the deamination of serine and threonine in the presence of an:nnonium sulfate-precipitated preparations follows a fairly straight line in the first few hours when plotted against time.
This observation does not provide an airtight proof of the nature of the system. Proteins and peptides can also do so by virtue of the proteolytic activities present in the preparations used. One is reminded of Braunstein and Asarkh's (76) experiments, where the oxidative deamination of certain amino acids in the presence of soil.
At first glance, one might try to explain the effect of pyridoxal phosphate on threonine deaminase as a function of... the activation of the conversion of threonine to alpha-aminobutyric acid by the same cofactor. The difficulties encountered in obtaining good resolution of the deaminases and pyridoxal phosphate are not uncommon among pyridoxal phosphate systems. In addition, the low deaminase activity may not be directly related to the pyridoxine-free nature of the mutant.
Analogies in the putative mechanisms of action of serine deaminase and cysteine desulfidrase and in the nature of the substrates prompted Glenn Fischer to test the effect of pyridoxal phosphate on Neurospora cysteine desulfhydrase. The acidic character of the alpha position in the amino acid part, which appears in step b, was demonstrated by Konikova et al. (96) with. incubation of alpha-deuterium amino acids with purified glutamic-pyruvic acid. Deuterium was not labilized during the reaction, leading the authors to assume that according to Schiff.
Blashko (110) has suggested that the enzymatic decarboxylation is mediated by the formation of a Schiff base between the amino acid substrate and pyridoxal phosphate, based on the observation that amino acids N-methyl.
C-COOH
3 -9- COOH
C_-COOH
R-C-COOH
It can be assumed that enzymatic decarboxylation takes place according to the rules of scheme 3, but with methyleneazo-. The similarity between enzymes and models is reinforced by the fact that enzymatic decarboxylation requires acidic pHs, whereas enzymatic transamination occurs only in basic media (100). In the above understanding of the mechanism of transamination and decarboxylation, there is a bond shift in the methyleneazometine bridge caused by conjugation with.
It is not difficult to see how the same shift can lead to deamination of serine, threonine and cysteine in the right catalytic environment. The three deaminations are assumed to occur via dehydration or desulfhydration to alpha-aminoacrylic acid or, in the case of threonine, to. In the case of cysteine, the existence of enzyme preparations that release hydrogen sulfide, but not.
Already in 1925, Bergman et al suggested that alpha-aminoacrylic acid stands at the crossroads of the biological interplay of serine, pyruvate, and alanine, studying as a model for those processes the nonenzymatic transformations undergone by glycylserine. Because of the inactivity of serine deaminase toward serine esterified through its hydroxyl group, Cbargaff and Sprinson (102) suggested the mediation of alpha-aminoacrylic acid. They incubated cyste ine and cyste ine de sulfhyclrase in the presence of labeled hydrogen sulfide.
It was found that the heavy sulfur content of cysteine was about 1% of that expected if it had reached equilibrium with the pool. The breakdown of tryptophan into indole, py1~1vat and ammonia can be described by the same scheme that replaces.
HOJ\-
COMPARISON OF' THE ACTIVITY OF' CALCIUM PYRIDOXAL PHOSPHATE AND OF PYRIDOXAL PHOSPHORIC ACID,
The slightly lower activity of the "clear" solution may be due to age or lower cofactor concentration. The stability of the calcium salt in phosphate buffer (pH 7.8) appears here greater than stated in the circular, which Merck & Co. sender with the samples of pyridoxal phosphate. However, due to the magnitude of the errors that can affect the blank-corrected ammonia values in the B6-less mixtures, R-values half as large as those given are also compatible with the experimental results.
No significant activation occurs upon addition of any of the putative cofactors, either alone or in conjunction with pyridoxal phosphate; while this latter substance caused a fivefold increase in deamination. When a solution of calcium pyridoxal phosphate (buffered at pH 7.8) is mixed with zinc sulfate, a white precipitate separates. They were recrystallized once from ethyl acetate, and the melting points were compared with those of synthetic samples of the 2,4-dinitrophenylhydrazones of pyruvic acid and alpha-tobutyric acid, respectively (Table XXIX).
The samples were brought to room temperature, warmed to about 20° .. below the expected melting point for about 15 minutes, and warmed for a final 10°. Control incubations were performed with comparable concentrations in 1.1 ml volumes of the reaction mixture with each of the following substrates: alpha-aminobutyric acid, alpha-ketoglutaric acid and no substrate (negative control). The occurrence of transamination in the test mixture was confirmed by paper chromatographic analysis.
Carbon dioxide production in the controls may be due to alpha-ketobutyrate or other compounds that could be decarboxylated (eg, pyruvate). The values from the manometric test correspond to 13% (or 11% if the readings in the controls are subtracted) of the theoretical yield, calculated on the assumption that each mole of alpha-ketoglutaric acid present results in one mole of alpha-ketobutyric acid. Extraction of the acidified test mixture with ether and further work-up of this extract did not yield alpha-ketobutyric acid.
Fraction 2, cooled in ice water, solidified mostly forming flame-like crystals like those of alpha-ketobutyric acid.
ADDITIONAL LITERATURE REVIEW
