Protein structure for support Shiff's base reactivity of transamination reaction.
| Project/Area Number |
06680619
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional biochemistry
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
TANASE Sumio KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE,ASSISTANT PROFESSOR, 医学部, 助教授 (20112401)
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| Co-Investigator(Kenkyū-buntansha) |
HIGAKI Tsuyosi KUMAMOTO UNIVERSITY COLLEGE OF MEDICAL SCIENSE,ASSISTANT PROFESSOR, 医療技術短期大学部, 助教授 (70128304)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Protein engineering / Aminotransferase / Pyridoxal / Catalytic activity / Enzyme structure / Aspartic acid / Amino acid substitution / NMR / タンパク質工学 / 部位特異的突然変異 / アミノ基転移 / 補酵素 / 核磁気共鳴 / ヒスチジン残基 / シッフ塩基 |
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| Research Abstract |
Porcine cytosolic aspartate aminotransferase (cAspAT) is composed of two identical subunits and has 8 histidyl residues in each subunit. Recent X-ray crystallographic studies show three histidyl residues (His143, His189 and His193) form a cluster below the bound coenzyme They are conserved in all AspATs so far examined. To clarify the role of this cluster, we prepared those histidine mutant enzymes to examine the effect on the environment of active site.
1.His143, His189 and His193 are replaceable to Gln residue without affecting the catalytic reactivity.
2.H189Q mutant enzyme increases both pK_a value of the internal aldimine and pK_1 value of the catalytic reaction by about 1 pH unit.
3.Substitution of His with Gln does not influence net charge of the enzyme, but the state of Schiff base does.
4.His189 proton signals are assigned on the ^1H NMR and His143 and His193 mutations change the chemical shift of His189 signals.
5.His -> Gln substitutions do not cause global conformation changes of the enzyme and the side chain of Gln residue occupies the position where His residue did (A.Arnone et al., personal communication).
6.pK_a values of His189 imidazole ring are similar to the pK_a value of internal aldimine observed by ^<13>C NMR studies in the absence/presence of succinate.
7.Peptide stretch, His193-Asn194, is located near imidazole ring of His189. We suspect that substitution with Gln may restrict the movement of His193-Asn194 peptide stretch and change the interaction between Asn194 and PLP 03'.
8.His189 imidazole ring can serve as a probe for the change of ionic state of active site in the catalysis.
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Report
(3 results)
Research Products
(8 results)
