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Title Page 2

Abstract 5

Contents 6

List of Abbreviations 13

1.1. Inroduction 17

1.1.1. Purine metabolism 17

1.1.2. 5'-Nucleotidase and its physiological role in purine metabolism 29

1.1.3. Regulation of purine metabolism by a novel 5'-nucleotidase from Saccharomyces cerevisiae 39

1.1.4. Biochemical properties of PfISN1 45

1.1.5. Structure and allosteric regulation of PfISN1 47

1.2. Purpose of this study 59

2.1. Materials and Methods 60

2.1.1. Cloning and purification of ScISN1 60

2.1.2. Crystallization and structure determination 69

2.1.3. Enzyme-coupled assay for phosphate 79

2.1.4. Analysis of kinetic data 91

2.1.5. Inorganic phosphate determination 92

2.1.6. High-performance liquid chromatography (HPLC) 95

2.1.7. LC/MS 97

2.1.8. Nuclear magnetic resonance (NMR) measurements 98

2.1.9. Circular dichroism (CD) spectra 99

2.2. Results 100

2.2.1. Structure of inosine liganded ScISN1* 100

2.2.2. Structural features of the ScISN1*(D172A)-IMP complex 119

2.2.3. IMP-induced changes and intersubunit communication 138

2.2.4. Biochemical and kinetic analysis of ScISN1 144

2.2.5. Site-directed mutagenesis analysis of ScISN1 152

2.2.6. Regulatory effect of ATP and inosine on ScISN1 activity 168

Discussion 184

Conclusion 195

References 196

Accession numbers 203

Abstract in Korean 204

List of Tables 12

Table 1. List of primer sequences for the structural analysis of ScISN1 63

Table 2. List of mutagenic primers for the biochemical analysis of ScISN1 64

Table 3. Data collection and refinement statistics 76

Table 4. Kinetic parameters of ScISN1 toward IMP using two different assays 150

Table 5. Kinetic parameters of ScISN1 mutants targeting long range interaction... 158

Table 6. Kinetic parameters of ScISN1 mutants targeting intersubunit... 163

List of Figures 8

Introduction 8

Figure 1. Structure of the nucleotide, purines, and the initial steps of purine... 18

Figure 2. A scheme of purine nucleotide synthesis 20

Figure 3. Feedback inhibition of purine synthesis 23

Figure 4. A scheme of purine nucleotide catabolism and salvage pathway 25

Figure 5. A common catalytic mechanism of HAD phosphatases 30

Figure 6. Disorder-to-order transition of the critical helix 34

Figure 7. Comparison of active site upon ATP/IMP binding 36

Figure 8. The reaction of IMP phosphatase ISN1 in the context of purine... 41

Figure 9. Monomeric structure of PfISN1 48

Figure 10. Interface 1 and 2 in PfISN1 50

Figure 11. ATP binding site in the interface 1 52

Figure 12. Structural reorientation of the NTRD in PfISN1 upon IMP binding 55

Figure 13. A long-distance stacking interaction during IMP binding 57

Materials and Methods 8

Figure 14. Scheme of the constrcuts for ScISN1, ScISN1*, and ScISN1*(D172A) 61

Figure 15. Purification profiles and SDS-PAGE analysis of ScISN1* 67

Figure 16. Crystals of ScISN1*, ScISN1*-inosine complex and ScISN1*(D172A)-... 70

Figure 17. LC/MS analysis of purified ScISN1 73

Figure 18. Scheme of enzyme-coupled assay for steady-state kinetics assay of... 80

Figure 19. Phosphate depletion and ScISN1-dependent reaction 82

Figure 20. Measurement of ScISN1 enzyme activity using phosphate assay kit 84

Figure 21. Evaluation of potential interference of coupling enzyme components... 87

Figure 22. Standard curve of the enzyme-coupled assay at pH 8.0 and pH 6.5 89

Figure 23. Standard curve for inorganic phosphate by modified version of... 93

Results 9

Figure 24. Elution profile of size-exclusion chromatography of ScISN1 101

Figure 25. Monomeric structure of ScISN1* complexed with inosine 103

Figure 26. Intersubunit interface for tetramerization and dimerization 106

Figure 27. Bottom view of a dimer in the ScISN1*-inosine complex 108

Figure 28. N-terminal tails of ScISN1*-inosine complex 110

Figure 29. Sequence comparison of ScISN1 with homologs 112

Figure 30. Putative active site of ScISN1*-inosine complex 114

Figure 31. Inosine binding site of ScISN1*-inosine complex 117

Figure 32. Active site of ScISN1*(D172A) complexed with IMP 120

Figure 33. Phosphate in the active site of the ScISN1*(D172A)-IMP complex 122

Figure 34. Inosine-binding site in the ScISN1*(D172A)-IMP complex and... 124

Figure 35. Disorder-to-order transition of a loop β1-α6 in metal binding site 127

Figure 36. Disorder-to-order transition of a loop in the cap domain 129

Figure 37. The metal binding site and binding environment of IMP in the... 131

Figure 38. IMP-dependent changes in the active site of ScISN1*(D172A)-IMP... 133

Figure 39. The authentic IMP binding mode in the ScISN1*(D172A)-IMP... 136

Figure 40. Structural comparison of IMP-induced conformational changes in the... 139

Figure 41. Proposed long-range interaction network for intersubunit... 142

Figure 42. HPLC elution profile of an ScISN1-dependent reaction 145

Figure 43. Enzymatic properties of ScISN1 147

Figure 44. Specific activity of ScISN1 mutants at pH 6.5 153

Figure 45. CD spectra, SEC, and SDS-PAGE of ScISN1 enzymes 156

Figure 46. Kinetic analyses of ScISN1 mutants for proposed long-range... 161

Figure 47. Kinetic analyses of ScISN1 mutants for intersubunit interaction;... 164

Figure 48. Salt bridge for tetramerization and hydrophobic environment 166

Figure 49. Relative activity of ScISN1 with ATP and nucleosides 169

Figure 50. Effects of ATP on the PNPase activity 171

Figure 51. Inhibition of ScISN1 activity by ATP 173

Figure 52. Inhibition assay of ScISN1 with inosine 176

Figure 53. Initial velocity plots of ScISN1 in the absence of inosine by Chen's... 178

Figure 54. NMR analysis of ATP and inosine binding to ScISN1 by Prof. Jeong-... 181

Discussion 11

Figure 55. ATP-binding site in PfISN1 and the corresponding region in ScISN1 189

초록보기

 핵산 대사는 모든 생물체에서 세포 조절에 필수적인 역할을 한다. 퓨린 대사에서 이노신 일인산은 원핵생물에서 진핵생물에 이르기까지 중요한 분기점 역할을 한다. 흥미롭게도, 사카로미세스 세레비시아는 전형적인 퓨린 대사의 5'-핵산분해효소인 cytosolic 5'-nucleotiase Ⅱ (cN-Ⅱ) 를 결여하고 있으며, 대신 새롭게 발견된 5'-핵산분해효소 클래스인 IMP-specific 5'-nucleotiase 1 (ISN1) 에만 의존한다. cN-Ⅱ가 폭넓은 기질 특이성을 갖는 반면, 사카로미세스 세레비시아의 ISN1 (ScISN1)은 이노신 일인산에 대해 엄격한 기질 특이성을 보이며, 이를 퓨린 이화작용의 주요 중간체인 이노신으로 탈인산화한다. 본 학위 논문에서는 ScISN1의 결정구조, 생화학적 특성, 조절 메커니즘을 조사하여 이노신과 아데노신 삼인산에 의한 억제를 규명하였다. 구조 분석을 통해 이노신 일인산 결합이 국소적 영역의 구조 안정화와 서브 유닛 간 상호작용을 통한 전역적 구조 변화를 유도함을 확인했다. 생화학적 연구 결과, ScISN11은 아데노신 일인산-활성화 상동 효소인 말라리아 원충 유래 ISN1 (PfISN1)과는 대조적으로 기질에만 의존적인 구조적 변화를 나타냈다. PfISN1과의 서열 및 구조 비교를 통해 N-말단 및 C-말단 조절 요소에서의 주요 차이를 확인하였으며, 이는 기질 친화도와 효소 조절 메커니즘의 차이를 초래하는 것으로 제안할 수 있다. 이 연구는 ScISN1의 독특한 조절 메커니즘과 퓨린 대사에서 이노신 일인산 및 이노신 항상성 유지에서의 중요한 역할을 강조한다.

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