The first description of a metalloenzyme-mediated nonoxidative decarboxylation reaction was that catalyzed by alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) (Li, T.; Walker, A. L.; Iwaki, H.; Hasegawa, Y.; Liu, A. J. Am. Chem. Soc. 2005, 127, 12282-12290 and Liu, A.; Zhang, H. Biochemistry 2006, 45, 10407-10411). Here, we report a rapid kinetic study of the ACMSD reaction using the cobalt(II)-reconstituted enzyme. Two previously undetected intermediates of the reaction are characterized. These are proposed to be the aminomuconate-epsilon-semialdehyde (AMS) product of the decarboxylation in complex with the enzyme and the free AMS after release from ACMSD. It was possible to determine the rates of formation of the enzyme-substrate complex (2.4 x 10(6) s(-1) M-1), release of the AMS product (8.8 s(-1)), and spontaneous cyclization of AMS to picolinic acid (0.05 s(-1)), which is the final product of the reaction. Reconstructed spectra of these reaction intermediates and a kinetic mechanism for the overall reaction are presented.