R67, the Other Dihydrofolate Reductase:  Rational Design of an Alternate Active Site Configuration†

Biochemistry ◽  
2008 ◽  
Vol 47 (2) ◽  
pp. 555-565 ◽  
Author(s):  
Jian Feng ◽  
Sumit Goswami ◽  
Elizabeth E. Howell
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Rational Design ◽  
The Other

scholarly journals Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

2009 ◽  
Vol 417 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Melissa A. Vargo ◽  
W. Edward Martucci ◽  
Karen S. Anderson
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Reductase Activity ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Single Polypeptide Chain ◽  
Species Specific

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS–DHFR C. hominis reveals a dimer with a ‘crossover helix’, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an ‘alanine-face’ enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a ‘glycine-face’ enzyme in which the same residues were mutated to glycine, and an ‘all-alanine’ helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s−1, the glycine-face mutant enzyme was 17 s−1, and the all-alanine helix enzyme was 16 s−1, all substantially impaired from the wild-type DHFR activity of 152 s−1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS–DHFR that could be exploited as a target for species-specific non-active site inhibitors.

scholarly journals Flower-Shaped C-Dots/Co3O4{111} Constructed with Dual-Reaction Centers for Enhancement of Fenton-Like Reaction Activity and Peroxymonosulfate Conversion to Sulfate Radical

Catalysts ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 135
Author(s):  
Zhibin Wen ◽  
Qianqian Zhu ◽  
Jiali Zhou ◽  
Shudi Zhao ◽  
Jinnan Wang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
High Surface ◽  
High Surface Area ◽  
Reaction Centers ◽  
Organic Radicals ◽  
The Other ◽  
High Catalytic Activity ◽  
High Adsorption ◽  
High Turnover

Novel flower-shaped C-dots/Co3O4{111} with dual-reaction centers were constructed to improve the Fenton-like reaction activity and peroxymonosulfate (PMS) conversion to sulfate radicals. Due to the exposure of a high surface area and Co3O4{111} facets, flower-shaped C-dots/Co3O4{111} could provide more Co(II) for PMS activation than traditional spherical Co3O4{110}. Meanwhile, PMS was preferred for adsorption on Co3O4{111} facets because of a high adsorption energy and thereby facilitated the electron transfer from Co(II) to PMS. More importantly, the Co–O–C linkage between C-dots and Co3O4{111} induced the formation of the dual-reaction center, which promoted the production of reactive organic radicals (R•). PMS could be directly reduced to SO4−• by R• over C-dots. On the other hand, electron transferred from R• to Co via Co–O–C linkage could accelerate the redox of Co(II)/(III), avoiding the invalid decomposition of PMS. Thus, C-dots doped on Co3O4{111} improved the PMS conversion rate to SO4−• over the single active site, resulting in high turnover numbers (TONs). In addition, TPR analysis indicated that the optimal content of C-dots doped on Co3O4{111} is 2.5%. More than 99% of antibiotics and dyes were degraded over C-dots/Co3O4{111} within 10 min. Even after six cycles, C-dots/Co3O4{111} still remained a high catalytic activity.

scholarly journals Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

2011 ◽  
Vol 176 (1) ◽  
pp. 52-59 ◽  
Author(s):  
Vivian Cody ◽  
Jim Pace ◽  
Jennifer Piraino ◽  
Sherry F. Queener
Keyword(s):  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Active Site ◽  
Crystallographic Analysis ◽  
Human Dihydrofolate Reductase

scholarly journals Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme

2005 ◽  
Vol 391 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Olga E. Skirgello ◽  
Peter V. Binevski ◽  
Vladimir F. Pozdnev ◽  
Olga A. Kost
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzymatic Activity ◽  
Active Site ◽  
The Other ◽  
Converting Enzyme ◽  
Human Enzyme ◽  
Independent Functions ◽  
The Common ◽  
The Individual ◽  
Hydrolysis Of

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. σ=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals Correlations of Inhibitor Kinetics forPneumocystis jiroveciiand Human Dihydrofolate Reductase with Structural Data for Human Active Site Mutant Enzyme Complexes†‡

Biochemistry ◽  
2009 ◽  
Vol 48 (8) ◽  
pp. 1702-1711 ◽  
Author(s):  
Vivian Cody ◽  
Jim Pace ◽  
Jennifer Makin ◽  
Jennifer Piraino ◽  
Sherry F. Queener ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Structural Data ◽  
Mutant Enzyme ◽  
Human Dihydrofolate Reductase ◽  
Enzyme Complexes ◽  
Inhibitor Kinetics

Methotrexate analogs. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductase

1982 ◽  
Vol 25 (8) ◽  
pp. 960-964 ◽  
Author(s):  
A. Rosowsky ◽  
J. E. Wright ◽  
C. Ginty ◽  
J. Uren
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site

Role of the Active-Site Carboxylate in Dihydrofolate Reductase: Kinetic and Spectroscopic Studies of the Aspartate 26 .fwdarw. Asparagine Mutant of the Lactobacillus casei Enzyme

Biochemistry ◽  
1995 ◽  
Vol 34 (9) ◽  
pp. 2872-2882 ◽  
Author(s):  
J. Basran ◽  
M. G. Casarotto ◽  
I. L. Barsukov ◽  
G. C. K. Roberts
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Lactobacillus Casei ◽  
Spectroscopic Studies
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