A mutational analysis of the active site of human type II inosine 5′-monophosphate dehydrogenase

被引:19
作者
Futer, O [1 ]
Sintchak, MD [1 ]
Caron, PR [1 ]
Nimmesgern, E [1 ]
DeCenzo, MT [1 ]
Livingston, DJ [1 ]
Raybuck, SA [1 ]
机构
[1] Vertex Pharmaceut Inc, Cambridge, MA 02139 USA
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 2002年 / 1594卷 / 01期
关键词
inosine monophosphate dehydrogenase; site-directed mutagenesis; X-ray structure; mycophenolic acid; active site; mechanism of catalysis;
D O I
10.1016/S0167-4838(01)00277-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:27 / 39
页数:13
相关论文
共 36 条
  • [21] NAGAI M, 1991, CANCER RES, V51, P3886
  • [22] NAGAI M, 1992, CANCER RES, V52, P258
  • [23] NATSUMEDA Y, 1990, J BIOL CHEM, V265, P5292
  • [24] GENETIC AND BIOCHEMICAL STUDIES OF GUANOSINE 5'-MONOPHOSPHATE PATHWAY IN ESCHERICHIA COLI
    NIJKAMP, HJJ
    DEHAAN, PG
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA, 1967, 145 (01) : 31 - +
  • [25] Biochemical analysis of the modular enzyme inosine 5′-monophosphate dehydrogenase
    Nimmesgern, E
    Black, J
    Futer, O
    Fulghum, JR
    Chambers, SP
    Brummel, CL
    Raybuck, SA
    Sintchak, MD
    [J]. PROTEIN EXPRESSION AND PURIFICATION, 1999, 17 (02) : 282 - 289
  • [26] Rashtchian A, 1992, PCR Methods Appl, V2, P124
  • [27] The conformation of NADH bound to inosine 5′-monophosphate dehydrogenase determined by transferred nuclear overhauser effect spectroscopy
    Schalk-Hihi, C
    Zhang, YZ
    Markham, GD
    [J]. BIOCHEMISTRY, 1998, 37 (20) : 7608 - 7616
  • [28] TISSUE-DIFFERENTIAL EXPRESSION OF 2 DISTINCT GENES FOR HUMAN IMP DEHYDROGENASE (EC-1.1.1.205)
    SENDA, M
    NATSUMEDA, Y
    [J]. LIFE SCIENCES, 1994, 54 (24) : 1917 - 1926
  • [29] The structure of inosine 5′-monophosphate dehydrogenase and the design of novel inhibitors
    Sintchak, MD
    Nimmesgern, E
    [J]. IMMUNOPHARMACOLOGY, 2000, 47 (2-3): : 163 - 184
  • [30] Structure and mechanism of inosine monophosphate dehydrogenase in complex with the immunosuppressant mycophenolic acid
    Sintchak, MD
    Fleming, MA
    Futer, O
    Raybuck, SA
    Chambers, SP
    Caron, PR
    Murcko, MA
    Wilson, KP
    [J]. CELL, 1996, 85 (06) : 921 - 930