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The effective molarity (EM) for a fragment truncated from the substrate or for a substituted active-site side chain at the transition state for the whole enzyme-catalyzed reaction is equal to kcat\/Km (M\u20131 s\u20131) for the wild-type enzyme-catalyzed reaction divided by the rate constant (M\u20132 s\u20131) for the third-order enzyme-catalyzed reaction of the substrate or enzyme pieces. These EMs are the predicted concentration of the deleted substrate or enzyme piece in the third-order reaction required to give the reaction velocity observed for the wild-type enzyme-catalyzed reaction. The EMs determined for a variety of enzymatic reactions range from 4 to 109 M. These variations are interpreted using a model that assumes an \u22486 kcal\/mol advantage for the reaction of whole species that arises from the larger entropic barrier to the higher-order reactions of enzyme or substrate fragments. Interactions that specifically stabilize or destabilize the transition states for enzyme-catalyzed reactions of the fragments also affect the EM. Large EMs are observed when fragment binding to the enzyme is impaired, and small EMs result when the fragment complex is stabilized by interactions between second-shell active-site side chain(s). The reported EM values are largely entropically controlled, consistent with a high degree of structural organization at the active sites for the wild-type and variant enzymes.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00503","type":"journal-article","created":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T14:01:24Z","timestamp":1789740084000},"page":"3044-3056","source":"Crossref","is-referenced-by-count":0,"title":["Enzyme Catalysis:\nThe Effective Molarity for Fragments\nof Enzyme-Bound Whole Substrates or Active-Site Amino Acid Side Chains"],"prefix":"10.1021","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0440-2387","authenticated-orcid":true,"given":"John P.","family":"Richard","sequence":"first","affiliation":[{"name":"University at Buffalo, SUNY , , , ,","place":["Buffalo, New York, United States, 14260-3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,18]]},"reference":[{"key":"2026100608071621900_cit1","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1038\/161707a0","article-title":"The nature\nof forces between large molecules of biological interest","volume":"161","author":"Pauling","year":"1948","journal-title":"Nature"},{"key":"2026100608071621900_cit2","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1126\/science.3299704","article-title":"Engineering Enzyme Specificity by \u201cSubstrate-Assisted\nCatalysis\u201d","volume":"237","author":"Carter","year":"1987","journal-title":"Science"},{"key":"2026100608071621900_cit3","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1002\/pro.5560010111","article-title":"Bronsted analysis\nof aspartate aminotransferase via\nexogenous catalysis of reactions of an inactive mutant","volume":"1","author":"Toney","year":"1992","journal-title":"Protein Sci."},{"key":"2026100608071621900_cit4","doi-asserted-by":"publisher","first-page":"1485","DOI":"10.1126\/science.2538921","article-title":"Directed Bronsted analysis of the restoration of activity\nto a mutant enzyme by exogenous amines","volume":"243","author":"Toney","year":"1989","journal-title":"Science"},{"key":"2026100608071621900_cit5","doi-asserted-by":"publisher","first-page":"7525","DOI":"10.1021\/bi300472n","article-title":"Modification\nof Residue 42 of the\nActive Site Loop with a Lysine-Mimetic Side Chain Rescues Isochorismate-Pyruvate\nLyase Activity in Pseudomonas aeruginosa PchB","volume":"51","author":"Olucha","year":"2012","journal-title":"Biochemistry"},{"key":"2026100608071621900_cit6","doi-asserted-by":"publisher","first-page":"11089","DOI":"10.1021\/jacs.7b03547","article-title":"An Activator\u2013Blocker Pair\nProvides a Controllable On\u2013Off Switch for a Ketosteroid Isomerase\nActive Site Mutant","volume":"139","author":"Lamba","year":"2017","journal-title":"J. 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