TY - JOUR
T1 - Mechanism of the conformational transitions in 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase as revealed by NMR spectroscopy
AU - Li, Guangyu
AU - Felczak, Krzysztof
AU - Shi, Genbin
AU - Yan, Honggao
PY - 2006/10/17
Y1 - 2006/10/17
N2 - 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK⊙MgAMPCPP and the ternary complex HPPK⊙MgAMPCPP⊙DMHP, where α,β-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK⊙MgAMPCPP are significantly different from those of HPPK in complex with Mgβ,γ-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK⊙MgATP be represented by those of HPPK⊙MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.
AB - 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK⊙MgAMPCPP and the ternary complex HPPK⊙MgAMPCPP⊙DMHP, where α,β-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK⊙MgAMPCPP are significantly different from those of HPPK in complex with Mgβ,γ-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK⊙MgATP be represented by those of HPPK⊙MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.
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U2 - 10.1021/bi061057m
DO - 10.1021/bi061057m
M3 - Article
C2 - 17029412
AN - SCOPUS:33750050233
SN - 0006-2960
VL - 45
SP - 12573
EP - 12581
JO - Biochemistry
JF - Biochemistry
IS - 41
ER -