Plant succinic semialdehyde dehydrogenase: Dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy

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.