DOI: 10.1246/bcsj.60.3017. Protein Engineering of Dihydrofolate Reductase. Improved Catalytic Step of Mutant-Enzymes

Protein Engineering of Dihydrofolate Reductase. Improved Catalytic Step of Mutant-Enzymes

10.1246/bcsj.60.3017

Crossref journal-article

Oxford University Press (OUP)

Bulletin of the Chemical Society of Japan (286)

Abstract

Abstract Two site-specific mutations on dihydrofolate reductase from Escherichia coli have been carried out at the Phe-31 site. From the crystallographic structure the Phe-31 is located at the dihydrofolate binding site and interacts with both the pteridine ring and the p-aminobenzoyl moiety of the substrate. Two mutant enzymes (Phe-31→Tyr and Val) have been purified to homogeneity and characterized by steady-state kinetics. The two mutations are aimed at assessing the hydrophobic interaction between the phenyl ring and the aromatic ring moiety of the substrate. Despite the fact that the first mutation has introduced a polar Tyr-group into a hydrophobic binding site, the Michaelis constant KDHF (KM with saturating NADPH and varying dihydrofolate) has increased only five-fold. The second mutation (Phe-31 →Val-31) results in a 25-fold increase in KDHF. More importantly, the maximum velocity of both mutant enzymes has increased more than 100%, indicating that these are better enzymes under the condition of [substrate]&gt;KDHF. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.

Bibliography

Taira, K., Chen, J.-T., Mayer, R. J., & Benkovic, S. J. (1987). Protein Engineering of Dihydrofolate Reductase. Improved Catalytic Step of Mutant-Enzymes. Bulletin of the Chemical Society of Japan, 60(8), 3017â3024.

Authors 4

Kazunari Taira (first)
Jin-Tann Chen (additional)
Ruth J Mayer (additional)
Stephen J Benkovic (additional)

References 29 Referenced 1

10.1073/pnas.79.21.6409
10.1126/science.6356360
10.1002/anie.198404673
10.1126/science.3838593
10.1002/jcb.240290203
{'key': '2024012102071016300_r6'}
{'key': '2024012102071016300_r7'}
{'key': '2024012102071016300_r8'}
{'key': '2024012102071016300_r9'}
10.1093/nar/8.10.2255
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10.1016/S0076-6879(80)65059-9
10.1016/S0076-6879(74)34025-6
10.1021/bi00307a034
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10.1093/nar/7.8.2115
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10.1139/v84-038
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{'key': '2024012102071016300_r22'}
{'key': '2024012102071016300_r23'}
10.1021/ja00337a028
{'key': '2024012102071016300_r25'}
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{'key': '2024012102071016300_r29'}

Dates

Type	When
Created	19 years, 1 month ago (July 25, 2006, 9:45 p.m.)
Deposited	1 year, 7 months ago (Jan. 20, 2024, 9:07 p.m.)
Indexed	1 year, 7 months ago (Jan. 21, 2024, 12:01 a.m.)
Issued	38 years ago (Aug. 1, 1987)
Published	38 years ago (Aug. 1, 1987)
Published Online	19 years, 2 months ago (June 27, 2006)
Published Print	38 years ago (Aug. 1, 1987)

Funders 0

None

BibTeX

@article{Taira_1987, title={Protein Engineering of Dihydrofolate Reductase. Improved Catalytic Step of Mutant-Enzymes}, volume={60}, ISSN={1348-0634}, url={http://dx.doi.org/10.1246/bcsj.60.3017}, DOI={10.1246/bcsj.60.3017}, number={8}, journal={Bulletin of the Chemical Society of Japan}, publisher={Oxford University Press (OUP)}, author={Taira, Kazunari and Chen, Jin-Tann and Mayer, Ruth J and Benkovic, Stephen J}, year={1987}, month=aug, pages={3017–3024} }

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