Intermediary Metabolism of Diplococcus Glycinophilus

KLEIN, SIGRID MI. (Brigham Young University, Provo, Utah) AND RICHARD D. SAGERS. Intermediary metabolism of Diplococcus glycinophilus. II. Enzymes of the acetate-generating system. J. Bacteriol. 83:121-126. 1962-The enzymatic steps in the conversion of glycine to acetate by Diplococcus glycinophilus were examined and the reaction rates of the individual steps compared with the maximal glycine fermentation rate by whole cells. Glycine is oxidatively cleaved to one-carbon units with the alpha carbon being transferred to tetrahydrofolate at the oxidation level of formaldehyde. The activated one-carbon unit is condensed with a second glycine molecule to form serine which is then deaminated to pyruvate. Pyruvate gives rise to acetyl units as acetyl coenzyme A and acetyl phosphate. The latter compound reacts with adenosine diphosphate to yield acetate and adenosine triphosphate, providing the main energy-yielding reaction in the fermentation. The enzyme systems discussed together with their specific activities (,umoles substrate acted upon per hr per mg protein) are as follows: serine aldolase, 9 to 12; serine dehydrase, 180; pyruvate-oxidizing and acyl unitgenerating system, 96; phosphotransacetylase, 96; acetokinase, 3,600. The high activity of the acetokinase system may tend to pull the entire reaction series in the direction of acetate and adenosine triphosphate generation. In all cases the reaction rates of the individual enzyme systems were equal to or significantly greater than the over-all glycine fermentation rate by whole cells (9 ,umoles per hr per mg protein). If coupled together, these enzymes could account for the fermentation of glycine to acetate, C02, and ammonia and could account for the conversion of 2-labeled glycine to doubly-labeled acetate as demonstrated in previous tracer studies. In a previous communication (Sagers and Gunsalus, 1961), the oxidative cleavage of glycine to one-carbon units by Diplococcus glycinophilus' was described and co-factor requirements for this cleavage defined. A reaction sequence which would allow the fermentation of 2-labeled glycine to doubly labeled acetate was proposed. The present communication presents evidence to substantiate that proposed pathway by defining the individual enzymatic steps of the glycine to acetate fermentation and establishing a reaction rate for each of the enzyme systems involved. The individual enzyme rates are compared with the maximum rate of glycine fermentation by whole cells. MATERIALS AND METHODS Cultivation of cells and preparation of cellfree extracts of D. glycinophilus as well as methods for the determination of ammonia, formaldehyde, pyruvate, and tetrahydrofolate compounds have been described previously (Sagers and Gunsalus, 1961). The nitrogen content of whole cells was determined by micro-Kjeldahl methods using the indicator system described by Sobel, Mayer, and Gottfried (1944). Protein content of cell-free extracts was determined turbidimetrically by the trichloroacetic acid method of Stadman, Novelli, and Lipmann (1951), using crystalline bovine albumin as a protein standard. Acetyl coenzyme A was determined as the heatand acid-stable thioester (Stadtman, 1952) by the ferric hydroxamate method of Lipmann and Tuttle (1945). Acetyl phosphate was prepared from iso' Although Bergey's Manual refers to this organism as Peptococcus glycinophilus, the terminology used in this paper will follow that used in paper I of this series (J. Bacteriol. 81:541-549, 1961). 121 on O cber 7, 2017 by gest http/jb.asm .rg/ D ow nladed fom