Trapping of an IMP dehydrogenase-substrate covalent intermediate by mycophenolic acid

Journal of the American Chemical Society

Volumn 118, Issue 8, 1996, Pages 2091-2092

  Link, John O ^a   Straub, Kenneth ^a  

^a ROCHE BIOSCIENCE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

INOSINATE DEHYDROGENASE; MYCOPHENOLIC ACID;

ARTICLE; ENZYME BINDING; ENZYME INHIBITION; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; REACTION ANALYSIS;

EID: 0029670363     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja9534056     Document Type: Article

References (22)

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13. max of 248 nm, as is tjpical for C2-thioalkyl inosines; see: Wong, C. G., Meyer, R. B., Jr. J. Med. Chem. 1984, 27, 429-432. The mass measurements. UV spectrum, and requirement of NAD for the formation of T38* are consistent with the structure of T38* presented in Figure 1B corresponding to covalent intermediate 3 in Figure 2A. and are not consistent with the isolation of the tetrahedral intermediate 2 from Figure 2A
14. 331Cys, human type II IMPDH) has been identified that reacts with the affinity labeling agent 6-chloro-IMP; see: (a) Brox. L. W.; Hampton, A. Biochemistry 1968, 7. 2589-2596. (b) Antonino, L. C.; Straub, K.; Wu, J. C. Biochemistry 1994, 33, 1760-1765.
15. 331Cys, human type II IMPDH) has been identified that reacts with the affinity labeling agent 6-chloro-IMP; see: (a) Brox. L. W.; Hampton, A. Biochemistry 1968, 7. 2589-2596. (b) Antonino, L. C.; Straub, K.; Wu, J. C. Biochemistry 1994, 33, 1760-1765.
16. 3). treated with excess dithiothreitol and iodoacetamide. and digested with trypsin (7.5 mu;L. 0.4 mg/mL. 20 h. 37 °C). To demonstrate covalent intermediate formation in the uninhibited pathway, an experiment without MPA but otherwise identical to that above was performed. On-line HPLC-MS and tandem MS were carried out as in ref 9b
17. After our isolation and characterization of the IMPDH-IMP covalent intermediate 3 from human IMPDH, we communicated our results to Dr. John C. Wu. He subsequently collaborated with Wang et al. in repeating experiments analogous to ours but using Tritrichomonas foetus IMPDH and similarly observed covalent catalysis in the uninhibited turnover of this protozoan enzyme: see: Huete-Perez, J. A.; Wu. J. C.; Whitby, F. G.; Wang, C. C. Biochemistry 1995, 34, 13889-13894.
18. Litt, M. R.; Potter, J. J.; Mezey, E.; Mitchell, M. C. Anal. Biochemistry 1989, 179, 34-36.
19. 3) to the protein afforded two 150 mu;L homogeneous samples. Ultrafiltration of one sample and HPLC analysis again showed no NADH in the filtrate. Trypsinolysis and HPLC analysis of the remaining sample of denatured protein afforded a tryptic map with 85% T38* relative to 15% T38, demonstrating the expected presence of the E - S covalent intermediate.
20. Prior proposals that MPA inhibition is through binding to IMPDH: XMP are contradicted by the demonstrated binding of MPA predominantly to the E - S covalent intermediate; see: Hupe, D. J.; Azzolina, B. A.: Behrens, N. D. J. Biol. Chem. 1986, 261, 8363-8369.
21. Rosenberry, T. L Advances in Enzymology: Meister, A., Ed ; John Wiley and Sons: New York, 1975; Vol. 43, pp 103-218.
22. We thank Dr. Kurt Jarnagm and Natalie Saldou for Edman microanalysis. John Link thanks Eva Papp and Dr. John C. Wu for helpful instruction in enzymological technique and in supplying the IMPDH used in these studies and Dr. Ming Chen and Professor E. J. Corey for helpful discussion.