Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we 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 ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH 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 Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD+ and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD+. GTP competed with ATP. However, only ATP increased the dissociation 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 GABA shunt by ATP.