Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

Ruszczycky, Mark W.; Anderson, Vernon E.

doi:10.1016/j.jtbi.2006.06.022

Cited by 32 publications

(32 citation statements)

References 45 publications

(54 reference statements)

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“…4.1), the isotope effect on κ ij is a constant function for all x > 0 , and therefore we may simply refer to it as the constant * κ ij independent of x . These isotope effects reflect progression from E i to each activated complex downstream and in general do not represent the intrinsic isotope effects associated with each individual elementary reaction (Stein, 1981; Ruszczycky and Anderson, 2006; Tian, 1992; Lewis and Schramm, 2006; Cleland, 1982). …”

Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

“…Sensitivity indices for net rate functions can be expressed (Ray, 1983; Ruszczycky and Anderson, 2006) in terms of the familiar concepts (Northrop, 1981b,a; Cleland, 1982) of catalytic commitments; however, it can also be shown (Alvarez et al, 1991; Stein, 1981; Murdoch, 1981; Ray, 1983) that they are related to the reciprocals of the κ ij according to…”

Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

“…Therefore, the relative contribution of each isotope effect * κ ij to

{{}^{*}k}_{i}^{'} (x)

can readily be inferred from the difference in energy between each activated complex and the first enzyme species along the subsequence as shown in Fig. 1A (Ruszczycky and Anderson, 2006). Moreover, limits of the sensitivity indices follow the same rules as do the limits of perturbation effects on net rate functions (see Prp.…”

Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

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Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants

Ruszczycky

Liu

2017

Measurement and Analysis of Kinetic Isotope Effects

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Steady-state kinetic isotope effects on enzyme catalyzed reactions are often interpreted in terms of the microscopic rate constants associated with the elementary reactions of interest. Unfortunately, this approach can lead to confusion, especially when more than one elementary reaction is isotopically sensitive, because it forces one to consider the full catalytic cycle one step at a time rather than as a complete whole. Herein we argue that shifting focus from intrinsic effects to net rate constants and enzyme intermediate concentrations provides a more natural and holistic interpretation by which the effects of partial rate-limitation are more easily understood. In doing so, we demonstrate how the experimental determination of isotope effects on enzyme intermediate concentrations allows a direct determination of isotope effects on net rate constants. The chapter is divided into three main sections. The first outlines the basic theory and its interpretation. The second discusses an application of the theory in the study of the radical SAM enzyme DesII. The final section then provides the complete mathematical treatment.

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Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

“…Therefore, the relative contribution of each isotope effect * κ ij to

{{}^{*}k}_{i}^{'} (x)

Section: Modeling Steady-state Isotope Effectsmentioning

confidence: 99%

See 1 more Smart Citation

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants

Ruszczycky

Liu

2017

Measurement and Analysis of Kinetic Isotope Effects

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“…In principle, any mechanistic step that involves a labeled molecule may exhibit an isotope effect (41). Inspection of the proposed reductive half-reaction of CAOs reveals several steps that are likely to display an isotope effect.…”

Section: Dependence Of Apparent K Cat /K M and D (K Cat /K M ) On Solmentioning

confidence: 99%

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Heuts

Gummadova

Pang

et al. 2011

Journal of Biological Chemistry

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Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK a values that defined a bell-shaped plot of turnover number k cat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k cat /K m ) app on pH revealed a single pK a value (ϳ9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k cat /K m ) app over the pH range of 6 to 10 was observed with d 2 -benzylamine. Over the same pH range, the KIE on k cat was found to be close to unity. The unusual KIE values on (k cat / K m ) app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k cat,app (8.01 ؎ 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.Copper amine oxidases or semicarbazide-sensitive amine oxidases (CAOs/SSAOs 3 ; EC 1.4.3.21/EC 1.4.3.22) comprise a group of copper-dependent enzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant release of hydrogen peroxide and ammonia. (R is a phenyl moiety in case of benzylamine and a benzyl moiety in case of phenylethylamine.)From the viewpoint of structure and mechanism, copper-dependent amine oxidases have been researched extensively for several decades, but relatively little is known about their physiological function.Human vascular adhesion protein-1 (VAP-1) is a dimeric membrane bound and circulating protein that is encoded by the AOC3 gene and is expressed mainly in endothelial cells of blood vessels, adipocytes, and smooth muscle cells (1-3). VAP-1 is a protein comprising two structural domains: an adhesive domain that targets leukocytes for transmigration and an amine oxidase domain (2, 4). At sites of inflammation on the endothelial cell surface, VAP-1 is involved in the recruitment and extravasation of lymphocytes and neutrophils. VAP-1 activity generates important signaling compounds such as hydrogen peroxide (5, 6). SSAO activity is also involved in glucose transport in adipose cells (1...

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“…For this scheme, assuming the chemical step is irreversible (i.e., k 4 and reverse commitment, C r = 0), the V/K KIE, symbolized as T (V/K), is related to the equilibrium BIE ( T K eq ) and the intrinsic KIE ( T k 3 ) by Equation 6 (46,47): (6) where T k 1 is the intrinsic KIE for binding and C f is the forward commitment to catalysis, defined as k 3 /k 2 . In cases where C f is low, the T k 1 C f term in Equation 6 can be approximated within experimental error as C f , 4 yielding Equation 7:…”

Section: Intrinsic Kinetic Isotope Effects From V/k Kinetic Isotope Ementioning

confidence: 99%

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

et al. 2007

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The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition state analogues show His 257 within hydrogen-bonding distance to the 5′-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (K m /K i ) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His 257 serves an important role in the early stages of transition state formation. Whereas mutation of His 257 resulted in little variation in the PNP·DADMe-ImmH·SO 4 structures, His257Phe·ImmH·PO 4 showed distortion at the 5′-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies on the remote 5′-3 H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5′-3 H intrinsic KIE to −3%, −14%, and 7%, respectively, while the BIEs contributed 2%, 2%, and −2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state. Keywordspurine nucleoside phosphorylase; mutagenesis; binding isotope effect; kinetic isotope effect; transition state; binding distortion; transition state geometry; dynamics Determination of the transition state structure of enzymatic reactions allows for the design of tight binding transition state analogue inhibitors. These transition state mimics can subvert the energetics that govern formation of the enzymatic transition state to achieve binding orders of magnitude stronger than that of substrate. The transition state structures of enzymatic reactions have frequently been established by measuring kinetic isotope effects (KIEs 1 ) from the competitive reaction of isotopically labeled substrates. These isotope effects on k cat /K m , often † Supported by NIH Research Grant GM41916.* To whom correspondence should be addressed. vern@aecom.yu.edu; Telephone, (718) 430-2813; Fax, (718) . ‡ Current address: Department of Chemistry, Barnard College, 3009 Broadway, New York, NY 10027. § Current address: Center for Synchrotron Bioscience, Brookhaven National Laboratory, Upton, NY 11973.1 Abbreviations: KIE, kinetic isotope effect; V/K KIE, kinetic isotope effect on the second-order rate constant, k cat /K m ; BIE, binding isotope effect; HsPNP, human purine nucleoside phosphorylase; PDB, Protein Data Bank; ImmH, Immucillin-H; DADMe-ImmH, 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H; RMS, root mea...

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Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

Cited by 32 publications

References 45 publications

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants

Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

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