The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS)