Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Biochemistry ◽  
1994 ◽  
Vol 33 (33) ◽  
pp. 9945-9952 ◽  
Author(s):  
Takayuki Nakano ◽  
H. Trent Spencer ◽  
James R. Appleman ◽  
Raymond L. Blakley
Keyword(s):  
Dihydrofolate Reductase ◽  
Critical Role ◽  
Inhibitor Binding ◽  
Human Dihydrofolate Reductase

Kinetic investigation of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase

Biochemistry ◽  
1990 ◽  
Vol 29 (27) ◽  
pp. 6428-6436 ◽  
Author(s):  
Jiu Tsair Tsay ◽  
James R. Appleman ◽  
William A. Beard ◽  
Neal J. Prendergast ◽  
Tavner J. Delcamp ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Kinetic Investigation ◽  
Human Dihydrofolate Reductase

scholarly journals Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors

1998 ◽  
Vol 331 (2) ◽  
pp. 375-379 ◽  
Author(s):  
Irene YIALLOUROS ◽  
Stamatia VASSILIOU ◽  
Athanasios YIOTAKIS ◽  
Robert ZWILLING ◽  
Walter STÖCKER ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Critical Role ◽  
Fold Increase ◽  
Rational Basis ◽  
Inhibitor Binding ◽  
Parent Molecule ◽  
Transition State Analogue ◽  
Morphogenetic Protein ◽  
Binding Behaviour

A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1·s-1) and dissociation (koff = 4.1×10-6 s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-O-methyl, Ki = 14 µM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stöcker (1996) Nature Struct. Biol. 3, 671–675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique ‘tyrosine switch ’ in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (5) ◽  
pp. 1432-1440 ◽  
Author(s):  
William A. Beard ◽  
James R. Appleman ◽  
Shaoming Huang ◽  
Tavner J. Delcamp ◽  
James H. Freisheim ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Active Site Residue ◽  
Human Dihydrofolate Reductase

scholarly journals Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

2001 ◽  
Vol 48 (4) ◽  
pp. 903-916 ◽  
Author(s):  
W Nowak ◽  
V Cody ◽  
A Wojtczak
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Structural Changes ◽  
Energy Substitution ◽  
Energy Simulations ◽  
Detailed Computer ◽  
Human Dihydrofolate Reductase ◽  
Folate Cycle ◽  
Active Site Mutants

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

scholarly journals Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

Biochemistry ◽  
1990 ◽  
Vol 29 (35) ◽  
pp. 8063-8069 ◽  
Author(s):  
Shaoming Huang ◽  
James R. Appleman ◽  
Xuehai Tan ◽  
Paul D. Thompson ◽  
Raymond L. Blakley ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Cofactor Specificity ◽  
Human Dihydrofolate Reductase

scholarly journals Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis

1989 ◽  
Vol 264 (34) ◽  
pp. 20786-20795
Author(s):  
B.I. Schweitzer ◽  
S Srimatkandada ◽  
H Gritsman ◽  
R Sheridan ◽  
R Venkataraghavan ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Human Dihydrofolate Reductase
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