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The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor\u2013acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.<\/jats:p>","DOI":"10.1073\/pnas.0606976103","type":"journal-article","created":{"date-parts":[[2006,10,11]],"date-time":"2006-10-11T02:54:28Z","timestamp":1160535268000},"page":"15753-15758","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":160,"title":["Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase"],"prefix":"10.1073","volume":"103","author":[{"given":"Lin","family":"Wang","sequence":"first","affiliation":[{"name":"*Department of Chemistry, University of Iowa, Iowa City, IA 52242; and"}]},{"given":"Nina M.","family":"Goodey","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Pennsylvania State University, University Park, PA 16802"}]},{"given":"Stephen 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