K. Busch et al., Plant succinic semialdehyde dehydrogenase: Dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy, BIOCHEM, 39(33), 2000, pp. 10110-10117
Recent kinetic studies revealed distinct modes of inhibition of mitochondri
al Arabidopsis thaliana succinic semialdehyde dehydrogenase (Ar-SSADH1) by
AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanis
m of feedback regulation of the GABA shunt by the respiratory chain. Here w
e used two approaches to investigate the interaction of ATP with At-SSADH1.
Cofactor displacement studies based on the reduced fluorescence intensity
of free NADH versus that of enzyme-bound NADH revealed that both AMP and AT
P decreased NADPI-At-SSADH1 complex formation. The competitive inhibitor AM
P displaced all bound NADH, while ATP, a noncompetitive inhibitor, could no
t, even in great excess, release all NADK from its binding site. To assess
the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to
monitor nucleotide binding to immobilized At-SSADH1. For this, we used a S
trep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The
tagged enzyme was tightly and reversibly captured by StrepTactin, which wa
s covalently immobilized on a CM5 chip. The binding constants for NAD(+) an
d ATP were determined from titration curves and were in good agreement with
the constants obtained from enzyme kinetics. Surface plasmon resonance mea
surements confirmed that ATP binds to a site different from the binding sit
e for NAD(+). GTP competed with ATP. However, only ATP increased the dissoc
iation constant of NAD(+) from SSADH. This explains the reduced affinity of
NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic
kinetics, and supports our model of feedback regulation of SSADH and the GA
BA shunt by ATP.