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1.
Adv Exp Med Biol ; 431: 221-6, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9598063

RESUMO

1. The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced. The gene product, hPurH has been overexpressed in E. coli, purified to homogeneity and crystallized. 2. The human pur H gene lies on chromosome 2, between band q34 and q35. There is at least one intron of 278 bp near the 5' end. 3. Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5. The existence of a linker or interaction region between the catalytic domains remains to be established. 4. Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first. 5. The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2. 6. Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting. 7. A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits. Probable binding sites for AICAR and FAICAR can be identified on each monomer. 8. Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM. 9. The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding. Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266. These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.


Assuntos
Cromossomos Humanos Par 2 , Hidroximetil e Formil Transferases/genética , Complexos Multienzimáticos/genética , Nucleotídeo Desaminases/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Sítios de Ligação , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Hidroximetil e Formil Transferases/biossíntese , Hidroximetil e Formil Transferases/química , Cinética , Modelos Moleculares , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/química , Nucleotídeo Desaminases/biossíntese , Nucleotídeo Desaminases/química , Conformação Proteica , Nucleotídeos de Purina/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Ribonucleotídeos/metabolismo
2.
Cancer Res ; 57(6): 1116-23, 1997 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9067281

RESUMO

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/farmacologia , Guanina/análogos & derivados , Hidroximetil e Formil Transferases , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , 5,10-Metilenotetra-Hidrofolato Redutase (FADH2) , Aciltransferases/antagonistas & inibidores , Aminoidrolases/antagonistas & inibidores , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Glutamatos/química , Guanina/química , Guanina/farmacologia , Humanos , Metotrexato/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2) , Estrutura Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Pemetrexede , Fosforribosilaminoimidazolcarboxamida Formiltransferase , Fosforribosilglicinamido Formiltransferase , Ácido Poliglutâmico/farmacologia , Quinazolinas/farmacologia , Tetra-Hidrofolatos/farmacologia , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacos
3.
Biochim Biophys Acta ; 1295(1): 81-8, 1996 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-8679677

RESUMO

p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Transaminases/genética , Transaminases/metabolismo , Aminação , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Carbono-Nitrogênio Ligases , Temperatura Baixa , Análise Mutacional de DNA , Escherichia coli/genética , Genes Bacterianos/genética , Glutamina/metabolismo , Dados de Sequência Molecular , Mutação , Compostos de Amônio Quaternário/metabolismo , Transaminases/química , Transaminases/isolamento & purificação
4.
Cancer Treat Res ; 87: 197-223, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8886454

RESUMO

In summary, the problem of MTX resistance has been approached in a mechanistic fashion, based on the wealth of information generated over the years. To date, these strategies have produced several new classes of anticancer drugs, with a variety of anticipated and unanticipated mechanisms of action. Several of these have shown promising preclinical activity, and these are moving into more stringent testing in the clinic.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Aminopterina/análogos & derivados , Aminopterina/farmacologia , Animais , Resistência a Medicamentos , Humanos , Metotrexato/farmacocinética , Metotrexato/farmacologia , Timidilato Sintase/antagonistas & inibidores
5.
J Biol Chem ; 271(4): 2225-33, 1996 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-8567683

RESUMO

We report here the cloning and sequencing of the cDNA, purification, steady state kinetic analysis, and truncation mapping studies of the human 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase). These steps of de novo purine biosynthesis, respectively. In all species of both prokaryotes and eukaryotes studied, these two activities are present on a single bifunctional polypeptide encoded on the purH gene. The human purH cDNA is 1776 base pairs in length encoding for a 591-amino acid polypeptic (Mr = 64,425). The human and avian purH cDNAs are 75 and 81% similar on the nucleotide and amino acid sequence level, respectively. The Km values for AICAR and (6R,6S)10-formyltetrahydrofolate are 16.8 microM +/- 1.5 and 60.2 microM +/- 5.0, respectively, for the cloned, purified human enzyme. A 10-amino acid sequence within the COOH-terminal portion of human AICARFT/IMPCHase has some degree of homology to a previously noted "folate binding site." Site directed mutagenesis studies indicate that this sequence plays no role in enzymatic activity. We have constructed truncation mutants which demonstrate that each of the two enzyme activities can be expressed independent of the other. IMPCHase and AICARFT activities are located within the NH2-terminal 223 and COOH-terminal 406 amino acids, respectively. The truncation mutant possessing AICARFT activity displays steady state kinetic parameters identical to those of the holoenzyme.


Assuntos
Aciltransferases/genética , Hidroximetil e Formil Transferases , Nucleotídeo Desaminases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , Primers do DNA/química , DNA Complementar/genética , Formiltetra-Hidrofolatos/metabolismo , Expressão Gênica , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleotídeo Desaminases/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
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