Structural Determinants of Enzyme Binding Affinity: The E1 Component of Pyruvate Dehydrogenase from Escherichia coli in Complex with the Inhibitor Thiamin Thiazolone Diphosphate ²,‡ Palaniappa Arjunan, §,|,⊥ Krishnamoorthy Chandrasekhar, §,|,⊥ Martin Sax, §,⊥ Andrew Brunskill, §,| Natalia Nemeria, ⊥,@ Frank Jordan, @ and William Furey* ,§,| Biocrystallography Laboratory, Veterans Affairs Medical Center, P.O. Box 12055, UniVersity DriVe C, Pittsburgh, PennsylVania 15240, Department of Pharmacology, UniVersity of Pittsburgh School of Medicine, 1340 BSTWR, Pittsburgh, PennsylVania 15261, and Department of Chemistry, Rutgers UniVersity, Newark, New Jersey 07102 ReceiVed September 4, 2003; ReVised Manuscript ReceiVed December 21, 2003 ABSTRACT: Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg 2+ has been determined at 2.1 Å and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg 2+ complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP- dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis. PDHc 1 E1 is the initial member of the pyruvate dehydro- genase multienzyme complex, an assemblage that plays a pivotal role in cellular metabolism catalyzing the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA. E1 catalyzes the first step of the multistep process, using ThDP and Mg 2+ as cofactors. In all thiamin-dependent enzymes, the reaction is initiated by the formation of a covalent adduct between the substrate and cofactor through the C2 atom of the thiazolium ring, as seen in the reaction path for E1 (Figure 1). As would be expected, blocking this site, for example, by replacing the proton on C2 with an oxygen atom as in thiamine thiazolone diphosphate (ThTDP, Figure 1) or with a sulfur atom as in thiamine thiothiazolone diphosphate (ThTTDP), inactivates the enzyme (1, 2). The binding affinities of ThTDP or ThTTDP relative to that of the cofactor ThDP are vastly different for the various thiamin-dependent enzymes. Gutowski and Lienhard (1) and more recently Nemeria et al.(2) have shown that ThTDP exhibits a significantly stronger binding affinity (>1000-fold) for Escherichia coli PDHc E1 than does ThDP, based on the lower limit of K i (0.003 μM) for ThTDP with isolated E. coli PDHc E1, compared to the K d (1.58 μM) for ThDP. In contrast, ThTDP is not a particularly potent inhibitor of several other ThDP-dependent enzymes. ThTDP binds to E. coli pyruvate oxidase (POX) only ∼30 times more strongly (K d.ThTDP ) 0.20 μM) than does the cofactor ThDP (3). On the basis of the rate of release of ThTDP from the enzyme, Kluger et al.(4) observed that ThTDP binds only slightly ² Supported at Veterans Affairs Medical Center and University of Pittsburgh School of Medicine by NIH Grant GM-61791 and by the VA Merit Review Program (to W.F.) and at Rutgers University by NIH Grants GM-50380 and GM-62330 and the Rutgers University Busch Fund (to F.J.). ‡ Coordinates have been deposited in the Protein Data Bank as entry 1RP7. * To whom correspondence should be addressed. Telephone: (412) 683-9718. Fax: (412) 688-6945. E-mail: fureyw@pitt.edu. § Veterans Affairs Medical Center. | University of Pittsburgh School of Medicine. ⊥ These authors contributed equally to the success of this study. @ Rutgers University. 1 Abbreviations: PDHc, pyruvate dehydrogenase multienzyme com- plex; ThDP, thiamin diphosphate; ThTDP, thiamin thiazolone diphos- phate; ThDP complex, PDHc E1-ThDP-Mg 2+ complex; ThTDP complex, PDHc E1-ThTDP-Mg 2+ complex; LThDP, C2R-lactylthi- amin diphosphate; HEThDP, C2R-hydroxyethylthiamin diphosphate; 2-AcThDP, C2R-acetylthiamin diphosphate; E1, first enzymatic com- ponent of multienzyme complexes related to and including PDHc; POX, pyruvate oxidase; PDC, pyruvate decarboxylase; TRK, transketolase; PEG, polyethylene glycol; NCS, noncrystallographic symmetry; RMS, root-mean-square; BNLSLS, Brookhaven National Laboratory Syn- chrotron Light Source. 2405 Biochemistry 2004, 43, 2405-2411 10.1021/bi030200y CCC: $27.50 © 2004 American Chemical Society Published on Web 02/12/2004