Monitoring Active Site Alterations upon Mutation of Yeast Pyruvate Kinase Using 205Tl+ NMR

Abstract

Yeast pyruvate kinase (YPK) 1 (EC 2.7.1.4.0) is a tetrameric enzyme of identical subunits with a subunit molecular mass of 54.5 kDa (1). PK is a key regulatory enzyme in glycolysis that catalyzes the nearly irreversible reaction of phosphoenolpyruvate (PEP) and ADP to yield pyruvate and ATP. YPK has an absolute requirement for both monovalent and divalent cations, undergoes homotropic activation by PEP and M 2ϩ , and heterotropic activation by fructose 1,6-bisphosphate (FBP). Potassium is the physiologically important monovalent activator, but several other monovalent cations (Li ϩ , Naϩ , CH 3 -NH 4 ϩ ) can also activate PK (2-4).The refined x-ray structures of the YPK-K ϩ -Mn 2ϩ -phosphoglycolate and YPK-K ϩ -Mn 2ϩ -phosphoglycolate-FBP complexes have been solved at 3 Å resolution (5). Phosphoglycolate is a structural analog of the substrate PEP that lacks the C-3 carbon. From the x-ray data, the active site Thr-298 is in the correct orientation to serve as the proton donor to the C-3 of the enolate of pyruvate, the enzyme-bound intermediate (Fig. 1). The role of Thr-298 in the YPK-catalyzed reaction has been addressed by mutation of this residue to serine, alanine (6), and to cysteine.2 Studies of these mutants suggest that Thr-298 is not the proton donor to the enolate of pyruvate but that enzyme-bound water serves this function. Far-UV CD analysis indicates that none of the mutations cause any significant change in the secondary structure and that wild type and mutant YPK enzymes are folded into a similar, if not identical, structure. Physical and kinetic studies with the Thr-298 mutants of YPK indicate that the single amino acid mutations at the active site can trigger long range effects. These effects were observed at the FBP-binding site, located more than 40 Å away from the active site (5). The Thr-298 residue is about 6.5 Å from the enzyme-bound divalent cation and 8.9 Å from the monovalent cation at the active site ( Fig. 1) (5).All three Thr-298 mutants of YPK, T298S, T298C and T298A, showed altered k cat and k cat /K m,PEP values and altered kinetic cooperativity with PEP relative to wild type PK (6). It is possible that these alterations in kinetic parameters of the YPK Thr-298 mutants may, in part, be explained by structural changes at the active site as a result of the single amino acid mutations. Such alterations may be too subtle to be monitored by methods such as CD and fluorescence spectroscopy, which are sensitive to more gross changes in protein structures.When performing site-directed mutagenesis studies, the possibility of local structural alterations introduced upon mutation tends to be overlooked in the interpretation and assessment of overall properties and behavior of the resulting protein mutants relative to the wild type protein. The ability to address these questions is normally limited by lack of appropriate experimental tools. Yeast PK has an advantage in that it is amenable to such an analysis. Measurement of the internuclear interactions between the mono-and divalent cations, b...

Proton Donor in Yeast Pyruvate Kinase: Chemical and Kinetic Properties of the Active Site Thr 298 to Cys Mutant

“…Hence, replacement of the hydroxyl functional group with a thiol group on the residue at position 298 may result in conformational changes around the bound PEP substrate in the active site. The T298C mutation does introduce significant conformational alterations of the substrate and the cations at the active site of YPK, as measured by 205 Tl + NMR (25). The interaction of FBP with the fully ligated kinetic complex of YPK is also affected by the T298C mutation (Table 1).…”

Cation Binding in Na,K-ATPase, Investigated by 205Tl Solid-State NMR Spectroscopy

“…Hinton and coworkers have reported chemical shifts for complexed 205 Tl-(I) between -700 and 300 ppm (11) and chemical shift anisotropies of up to 855 ppm (12). Previous NMR studies have demonstrated that the chemical shifts observed by 205 Tl NMR may be powerful probes for the investigation of protein binding sites (13,14) and conformational changes (15,16). Furthermore, 205 Tl longitudinal relaxation times, T 1 , have been examined and used as a means to obtain structural information (17).…”

NMR-Active Nuclei for Biological and Biomedical Applications

“…When oxalate was used as the anion instead of carbonate, the 205 Tl NMR signals originating from the bound metal ion in the sites of ovotransferrin were shifted downfield and became almost degenerate [359]. Conformational changes in wild-type and mutant forms of yeast pyruvate kinase have been investigated by 205 Tl NMR [360,361]. 205 Tl NMR methods have also been developed for the characterisation of monovalent cation binding to nucleic acids, including the first 1 H- 205 Tl scalar couplings observed in a biological system [362].…”