Download Novel nucleotide diversity of succinate thiokinase

X39
622nd MEETING. LEICESTER
Novel nucleotide diversity of succinate thiokinase
T I M O T H Y M. J E N K I N S and P. D. J . WEITZMAN
Dcptirtmont of' Biochcwiistrj. Universi!,. of Buth.
C l r w r t o n Down. Buth BA2 7 A Y . U .K.
observed in the presence of both A D P and I D P a t saturating
levels (Table IA). Interestingly. although G D P is not utilized by either enzyme, it appears to inhibit the two STKs
differentially (Table IA). The I-STK shows a greater degree
of inhibition by G D P than does the A-STK, and this may
be due to the structural similarity between IDP and G D P .
In addition. the two STKs exhibit different pH profiles and
have different K , values for their respective nucleotides: K ,
(IDP) = 26.7 f 2.1 P M and K , (ADP) = 89.4
11.9~~.
The relative specific activities of the two STKs in B. niogutrrium were found to vary depending upon the stage of
growth and also upon the growth medium. O n nutrient
broth a 10-fold variation in the ratio of A-STK/I-STK
occurred between early-logarithmic and late-stationary
growth phases, and between different growth media at the
same stage of growth (Table 1 B). Along with the evidence
presented in Table IA, these results strongly suggest the
presence of two distinct nucleotide-specific STKs, the relative proportions of which differ according to physiological
conditions. The marked elevation of I-STK activity during
growth on succinate may reflect increased formation of
succinyl-CoA from succinate under these conditions. The
situation in Gram-positive bacteria may therefore resemble
that in animal tissues, in which it has been suggested that
A-STK may function in the energetic role of the citric acid
cycle, whereas the second STK may operate in the opposite metabolic direction, converting succinate to succinylCoA for biosynthesis (Jenkins & Weitzman, 1986). Investigations in progress are aimed at further exploration o f this
possibility.
The nucleotide specificity of succinate thiokinase (STK)
from a variety of sources has been studied (Palmer &
Wedding. 1966; McClellan & Ottaway, 1980; Weitzman &
Jaskowska-Hodges, 1982). Gram-negative bacteria can
utilize both adenine and guanine nucleotides (ADP/ATP
and G D P G T P ) on a single 'large' (tetrameric) enzyme. In
contrast, the utilization of these nucleotides by animal STKs
occurs on two distinct 'small' (dimeric) enzymes (Weitzman
ct t d . . 1986). Gram-positive bacteria were previously
thought to contain a single 'small' (dimeric) STK specific for
ADP/ATP. However. this communication presents evidence
for the existence of a second STK activity in Gram-positive
bacteria which is specific for inosine nucleotides (IDP/ITP).
In animal tissues, where both A-STK and G-STK activities are present. I-STK activity can be detected, but in
tissues with apparently only A-STK. no I-STK is measurable. Similarly. in Gram-negative bacterial STK, inosine
nucleotides may replace their guanine counterparts with
similar kinetic characteristics. In Gram-positive bacteria,
where n o ti-STK occurs, a n STK specific for inosine nucleotides has been found and this I-STK appears distinct from
the A-STK.
Bacteria were grown in nutrient broth a t 30°C or 37°C.
Btic~illus nicJgutrrium was also grown in minimal salts
medium, with 25 mM-succinate, glucose or glutamate as the
sole carbon source, to detect any changes in levels of A-STK
and I-STK activities. STK activity was assayed polarographically (Weitzman & Jaskowska-Hodges, 1982). Hp.1.c.
analysis of all the nucleotides confirmed their purity and
hence ruled out the possibility of the detection of activity
with a minor impurity. I-STK was detected in the following
Gram-positive bacteria: B. mc>gaterium, Bacil1u.s suhtilis,
Sttiph!.loc.oc.c,u.s uurws, Arthrohucter simples and Kurthiu
~ o p f i iPartial
.
additivity which has previously been used as
a criterion for distinct enzymes (Weitzman et ul., 1986) was
Jenkins, T. M. & Weitzman. P. D. J. (19x6) FEBS L e i / . 205. 215 218
McClellan. J . A. & Ottaway. J. H. (1980) Comp. B i o h m . P l ~ ~ i o l .
678, 697 6x4
Palmer. J. M . & Wedding. R. T. (1966) Biochini. B i ~ p h x . Actu
~.
113.
167 174
WcitLman. 1'. D. J . & Jaskowska-Hodges, H. (19x2) FEBS Lrir. 143.
237 240
Wcit~man.P. D. J . . Jenkins. T. M.. Else. A. J . & Holt. R. A. (1986)
F'ERS L r i i . 199. 57 60
Abbreviations used: STK. succinate thiokinase; Hp.1.c.. high pressure liquid chromatography; NDP. nucleoside diphosphate.
Rcceived I April 19x7
Table I . Succinate thiokinase activities ,from Bacillus mcguterium
A
Nutrient broth
Late-stationary
+ADP
+IDP
+GDP
74.7
16.12
0.0
+(ADP
+ IDP)
+(ADP
80. I
+ GDP)
58.2
/n
'
Inhibition
+(IDP
22.2
+ GDP)
7.27
B
A-STK
I-STK
Ratio A-STKLSTK
Nutrient broth
Early-logarithmic
Mid-logarithmic
Late-stationary
31.0
73.3
48.74
0.35
4.8
5.56
xx.57
15.27
8.77
25 mM-Succinate
Late-stationary
41.09
6.75
6.1
25 mM-Glucose
Late-stationary
57.9
I .04
55.67
25 mM-Glutamate
Late-stationary
28.06
0.426
65.87
Growth media
(NDP
Vol. I5
=
0.5mM; all activity values are in nmol/min per mg protein.)
Yn Inhibition
54.8