Binding Energy and Catalysis:  The Implications for Transition-State Analogs and Catalytic Antibodies

Mader, Mary M.; Bartlett, Paul A.

doi:10.1021/cr960435y

Cited by 284 publications

(235 citation statements)

References 140 publications

(275 reference statements)

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“…Bartlett has therefore argued that a more appropriate criteria for assessing transition state analogy is that the relative affinity of an enzyme for the chemical transition state (given by k cat /K m ) and for a putative transition state analogue (given by 1/K i ) be constant over a range of values. 7,8 In other words, Δlog(K m /k cat ) = ΔlogK i .…”

Section: Introductionmentioning

confidence: 99%

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Messmore

Raines

2000

J. Am. Chem. Soc.

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Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2′,3′-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10 5 -fold in their k cat /K m values for catalysis, but <40-fold in their K i values for inhibition of catalysis by U>v. Plots of logK i vs log(K m /k cat ) for three distinct substrates [poly(cytidylic acid), uridine 3′-(p-nitrophenyl phosphate), and cytidine 2′,3′-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K i for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.

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Section: Introductionmentioning

confidence: 99%

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Messmore

Raines

2000

J. Am. Chem. Soc.

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“…The K i values of the acarviosyl-maltooligosaccharides AC5-AC10 for SBG were significantly lower compared with AC4, indicating that these acarviosyl-maltooligosaccharides are more effective inhibitors of SBG than AC4. The clear correlation observed between log K i for AC4ϪAC7 and log(K m /k cat ) for the hydrolysis of Glc 4 ϪGlc 7 suggests that the inhibitors are transition state analogs (29). Relative to the substrate, the tighter binding of the acarviosyl-maltooligosaccharides to the active site results in a value of K i , which is 3 orders of magnitude lower than K m (upon equating K m with K s ); this could be considered a consequence of the valienamine unit, which is considered to mimic the glycosyl cation-like transition state.…”

Section: Discussionmentioning

confidence: 98%

“…, where d and k non represent proportionality and non-enzymatic reaction rate constants, respectively (29). (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Structural Advantage of Sugar Beet α-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

Tagami

Yamashita²,

Okuyama

et al. 2015

Journal of Biological Chemistry

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Background: Most plant ␣-glucosidases prefer long-chain substrates. Results: Inhibitory and structural analyses using the unique 4 -10-mer inhibitors identified the substrate binding mode of the enzyme far from the active-site pocket. Conclusion:The structure of the substrate-binding subsites was suitable for single helical conformation of amylose. Significance: The enzyme seems to ingeniously use the self-stabilizing property of the substrate to form a stable ES complex.

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“…1) can be performed before converting substrates into inhibitors because transition-state theory for enzyme-catalyzed reactions predicts that the inhibitory activity (K i ) of mechanism-based inhibitors can be correlated with the inverse of the catalytic efficiency of the corresponding substrates (K m /k cat ) 10 . Other researchers have confirmed this correlation for peptide substrates 11,12 , and we have observed this correlation for non-peptidic substrates and inhibitors for both cathepsin S and chymotrypsin [1][2][3] .…”

Section: Introductionmentioning

confidence: 99%

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

2007

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Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates. INTRODUCTIONWe have developed the first substrate-based fragment identification method, called 'substrate activity screening' (SAS) 1-3 . This method addresses two key challenges in fragment-based screening: (i) the accurate and efficient identification of weak binding fragments and (ii) the rapid optimization of the initial weak binding fragments into higher-affinity compounds 4,5 . In this method (Fig. 1), a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl fragments is prepared and then, in a first step, the library is screened to identify protease substrates using a single-step, highthroughput fluorescence-based assay. The substrate-based screening method in Step 1 has important attributes in detecting weak binding fragments in addition to being high throughput and straightforward to perform. False positives as a result of aggregation, protein precipitation or non-specific binding seen in traditional inhibitor screens are not observed. In fact, in the present approach, both an active enzyme and productive active-site binding are required for the protease-catalyzed amide bond hydrolysis to occur and for the subsequent release of the fluorescent coumarin group to be observed. In addition, in contrast to direct binding assays and traditional inhibitor screens, catalytic substrate turnover results in signal amplification, and therefore even very weak substrates can be identified at concentrations where only minimal binding to the enzyme occurs. The second and third steps (Fig. 1), which are not covered in this protocol, provide a strategy for systematically and efficiently optimizing substrates incorporating weak binding fragments into high-affinity inhibitors. In the second step, the activity of the substrates is rapidly optimized by the straightforward solidphase synthesis and subsequent assay of focused libraries of substrate analogs. The

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Binding Energy and Catalysis: The Implications for Transition-State Analogs and Catalytic Antibodies

Cited by 284 publications

References 140 publications

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Structural Advantage of Sugar Beet α-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

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