Role of 5'-nucleotidase in thiopurine metabolism: enzyme kinetic profile and association with thio-GMP levels in patients with acute lymphoblastic leukemia during 6-mercaptopurine treatment.
Thiopurines are used for treatment of several diseases. Cytotoxicity is caused by the derived compounds 6-thioguanine nucleotides (TGNs) and methyl-6-thioinosine monophosphate (methylthio-IMP). The 6-thiopurine mononucleotides 6-thio-IMP (thio-IMP), 6-thio-GMP (thio-GMP) and methylthio-IMP can be catabolized by purine 5'-nucleotidase. It has been shown that the various 5'-nucleotidases are key enzymes for (6-thio)-purine metabolism. We aimed to investigate whether the overall 5'-nucleotidase (5'NT) activity is correlated with the efficacy and toxicity of 6-thiopurine nucleotides. Substrate affinity of 5'NT for IMP, GMP, AMP, thio-IMP, thio-GMP and methylthio-IMP was studied in human lymphocytes. For each of the substrates, the pH for optimal overall enzyme activity has been determined at a pH range between 6 and 10. At the optimal pH, assays were performed to establish Km and Vmax values. Optimal pH values for the various substrates were between 7 and 8.5. Km values ranged from 33 to 109 microM, Vmax ranged from 3.99 to 19.5 nmol/10(6) peripheral mononuclear cells (pMNC) h, and Vmax/Km ratios ranged from 105 to 250. The results did not show a distinct preference of 5'NT activity for any of the tested thiopurine nucleotides. The enzyme kinetic studies furthermore revealed substrate inhibition by thio-IMP and thio-GMP as a substrate. Inhibition by thio-GMP also seems to occur in patients treated with 6-mercaptopurine (6 MP); subsequently, this may lead to toxicity in these patients.
Department of Pediatrics, Division of Hemato-Oncology, University Medical Center St Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Connie
C
Brouwer
Trude M
TM
Vogels-Mentink
Jenneke J
JJ
Keizer-Garritsen
Frans J M
FJ
Trijbels
Jos P M
JP
Bökkerink
Peter M
PM
Hoogerbrugge
Elisabeth R
ER
van Wering
Anjo J P
AJ
Veerman
Ronney A
RA
De Abreu
D015720:5'-Nucleotidase / Q000378:metabolism*; D002648:Child; D002851:Chromatography, High Pressure Liquid; D006157:Guanosine Monophosphate / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015122:Mercaptopurine / Q000494:pharmacology / Q000627:therapeutic use*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013866:Thioguanine / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D006157:Guanosine Monophosphate; D015122:Mercaptopurine; D015720:5'-Nucleotidase; D013866:Thioguanine