التفاصيل البيبلوغرافية
العنوان: |
Roles of His205, His296, His303 and Asp259 in catalysis by NAD + ‐specific D‐lactate dehydrogenase |
المؤلفون: |
Kochhar, Sunil, Lamzin, Victor S., Razeto, Adelia, Delley, Michele, Hottinger, Herbert, Germond, Jacques‐Edouard |
المصدر: |
European Journal of Biochemistry ; volume 267, issue 6, page 1633-1639 ; ISSN 0014-2956 1432-1033 |
بيانات النشر: |
Wiley |
سنة النشر: |
2000 |
المجموعة: |
Wiley Online Library (Open Access Articles via Crossref) |
الوصف: |
The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD + ‐specific d ‐lactate dehydrogenase, was investigated using site‐directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because K m for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125‐fold reduction in k cat and a slight lowering of the K m value for pyruvate. D259N mutant showed a 56‐fold reduction in k cat and a fivefold lowering of K m . The enzymatic activity profile shifted towards acidic pH by ≈ 2 units. The H303K mutation produced no significant change in k cat values, although K m for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in k cat values or in K m for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the d ‐specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism. |
نوع الوثيقة: |
article in journal/newspaper |
اللغة: |
English |
DOI: |
10.1046/j.1432-1327.2000.01155.x |
الإتاحة: |
https://doi.org/10.1046/j.1432-1327.2000.01155.xTest |
حقوق: |
http://onlinelibrary.wiley.com/termsAndConditions#vorTest |
رقم الانضمام: |
edsbas.4D02748A |
قاعدة البيانات: |
BASE |