Table Of ContentTHE MECHANISM OF D-AMINO ACID FORMATION:
AN ALANINE RACEMASE FROM
STREPTOCOCCUS FAECALIS
BI
WILLIS AVERY WOOD
Bachelor of Science, 1947
Cornell University
Submitted to the Faculty of the Graduate School
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
in the Department of Bacteriology,
Indiana University,
February, 1950
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ACKNOYfLEDGMENT
The author takes this opportunity to express his gratitude to
Professor I. C. Gunsalus for the guidance and assistance so generously
rendered him. His scientific enthusiasm has furnished inspiration and
his valuable criticism and constant interest, both academic and per
sonal, has contributed greatly to the direction and execution of this
work.
VITA
Willis Avery Wood was born on August 6, 1921 in Johnson City,
New York# He attended the public schools of Binghamton, New York,
graduating from North Senior High School in 1940. He attended Cornell
University from 1940 to 1943> at which time he was ordered to active
duty in the Army of the United States, serving in the Quartermaster
Corps. In 1946 he was relieved of active duty and re-entered Cornell
University where he received the degree of Bachelor of Science in 1947*
He attended Indiana University from 1947 to 1950 as a graduate student
in Bacteriology. He is a member of the Society of American Bacteriolo
gists, American Chemical Society, and Sigma Xi.
iii
TO ALICE JANE
iv
table; of contents
Page
INTRODUCTION ................................................................................................................... 1
METHODS ........................................................................................................................... 4
RESULTS........................................................................................................................... 10
Preparation of a cell-free racemase .................................................. 11
Characteristics of alanine racemase .................................................. 12
MECHANISM....................................................................................................................... 15
DISTRIBUTION OF ALANINE RACEMASE................................................................... . 20
DISCUSSION..................................................................................................................... 22
SUMMARY........................................................................................................................... 26
BIBLIOGRAPHY.................................................................................................................. 29
OTHER STUDIES OF MICROBIAL AMINO ACID METABOLISM (reprints appended)
The Activity of Pyridoxal Phosphate in Tryptophane Formation
by Cell-Free Enzyme Preparations
Function of Pyridoxal Phosphate: Resolution and Purification
of the Tryptophanase Enzyme of Escherichia coli
Serine and Threonine Deaminases of Escherichia coli: Acti
vators for a Cell-Free System
v
INTRODUCTION
The literature on the general occurrence, the essentiality, and
the role of D-amino acids in biological systems do not support a unified
viewpoint. Reports of the relationship of D-amino acids to living systems
have appeared without an adequate knowledge concerning the importance,
occurrence, and formation of these compounds being reached. The data as
well as their interpretation are conflicting in many instances. Recent
studies, largely with bacteria, have served to re-emphasize the importance
of D-amino acids and have prompted the present examination of the mode of
formation of D-alanine.
K8gl and Erxleben (l) increased greatly the interest in D-amino
acids by their report of high D-glutamic acid content in malignant tissue.
As high as 15 to 45 per cent of the glutamic acid isolated was reported to
be the D-isomer. Later investigators failed to confirm this report although
several, including Ghibnall and co-workers (2), Graph, Rittenberg, and Fos
ter (3), and Wieland and Paul (4) > have found from 1 to 5 per cent of the
glutamic acid isolated from normal and malignant tissue to be of the D-
form. These investigators, however, attributed the presence of D-glutamic
acid to racemization during isolation and therefore attached little or no
importance to its presence.
In studies of microorganisms, D-amino acids have been reported in
extracellular material, as capsules or in the medium after growth. The
capsule of Bacillus anthracis (5* 6), and soluble polypeptides secreted
by Bacillus subtilis and Bacillus mesentericus (5) have been shown to be
composed exclusively of D-glutamic acid. More recently D-amino acid resi
dues have been reported in several antibiotics including gramicidin (7* £),
tyrocidin (9, 10), polymyxin (ll), aerosporin (12), circulin (13)> and
penicillin (14).
D-amino acids have also been reported as substrates for bacterial
growth (15-22). In the majority of cases, either the specificity of the
B-isomer was not shown or the L-isomer or keto acid analog permitted an
equal or greater rate of growth. In this type of experiment, Snell and
Guirard (23) reported DL-alanine to replace vitamin for the growth of
Streptococcus faecalis. strain R. Later, Snell (24) showed D-alanine to
be specifically required and not replaceable with the Ir-isomer. The D-
alanine, however, was replaceable by vitamin B^. In line with the general
hypothesis that an essential metabolite not required for growth is formed
by the organism during growth, Snell and Guirard (23) suggested that D-
alanine functions as a precursor for the formation of vitamin B.. How-
6
ever, Bellamy and Gunsalus (25) questioned the validity of this hypothesis
because cells grown in the presence of D-alanine were nearly devoid of co
decarboxylase, although they contained the tyrosine apodecarboxylase which
could be activated by vitamin B^ in the form of pyridoxal. Thus the alter
nate possibility, a function of vitamin B^ in the formation of D-alanine,
was indicated. A second instance of apoenzyme formation during growth in
a B deficient medium was reported for the transaminases by Lichstein,
o
Gunsalus, and Umbreit (26).
More recently, Holden, Furman and Snell (27)9 and Holden and Snell
(28) have confirmed the conclusions reached in the enzyme experiments. The
analysis, by microbiological assay, of cells grown in the presence of D-
alanine as replacement for vitamin B^ showed appreciable amounts of D-alanine
and negligible amounts of the vitamin, whereas cells grown with minimal B^
and no D-alanine contained appreciable amounts of both vitamin B^ and D-
alanine. This demonstration of a D-alanine requirement for growth and its
3
incorporation into cell material represents the first report of a D-amino
acid per se acting as an essential metabolite.
The demonstration that vitamin functions in the formation of D-
alanine opens the way for enzyme studies of the reactions involved. A con
sideration of possible routes of D-amino acid formation has suggested enzy
matic racemization and transamination (2B) as likely possibilities* In the
case of racemization, the D-amino acid could arise by inversion,of the L-
isomer, whereas in transamination it could be formed from pyruvate with
another amino acid serving as the amino group donor.
In the present study, D-alanine was found to arise by enzymatic
racemization with vitamin in the form of pyridoxal phosphate serving as
the coenzyme.
4
METHODS
Bacteriological: Streptococcus faecalis. strain R (ATCC #8043), previously
employed for studies of tyrosine decarboxylase (29), and glutamic-aspartic
transaminase (26) was used. Active racemase preparations were obtained by
growing the cells in medium AC.3, which contains 1 per cent each of yeast
extract and tryptone, 0,5 per cent dipotassium phosphate and 0*3 per cent
glucose. To obtain a large quantity of cells, 10 liter batches were grown
in 2g-gallon reagent bottles. The medium was inoculated with a 0.1 per
cent of an 8 to 12-hour culture and incubated 10 to 12 hours at 37° (final
pH, 5*6 to 5*S). The cells were harvested with a Sharpies centrifuge, washed
once with water by resuspending in one-tenth the growth volume and centri
fuging. The washed cells were either acetone dried or dried in vacuo to
yield lyophilized preparations*
Vacuum dried cells were prepared by suspending the washed cells in
water to about 10 per cent by weight and drying in vacuo over Drierite to
yield about 10 grams of dry cells per 10 liters of medium. Acetone dried
cells were prepared by pipetting a 10 per cent suspension of cells into 10
volumes of ice-cold acetone with rapid stirring. The cells were collected
on a buchner filter, washed with acetone and ether; and air dried. These
preparations racemized 50 to 150 microliters (jul) of alanine per mg dry wt
per hour and were stable for several weeks if kept dry or if stored at -20°.
One micromole (juM) of alanine is equivalent to 22.4 p i,
Chemical: D-alanine Determination. D-alanine was determined manometrically
using a partially purified D-amino acid oxidase (30) • Racemization of ala
nine was carried out in 16 mm test tubes using a 3 ml reaction volume
