Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme–reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

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Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805416115 ◽

2018 ◽

Vol 115 (27) ◽

pp. E6209-E6216 ◽

Cited By ~ 12

Author(s):

Rajesh K. Harijan ◽

Ioanna Zoi ◽

Dimitri Antoniou ◽

Steven D. Schwartz ◽

Vern L. Schramm

Keyword(s):

Transition State ◽

Purine Nucleoside Phosphorylase ◽

Isotope Effects ◽

Catalytic Site ◽

Path Sampling ◽

Transition Path ◽

Transition Path Sampling ◽

Steady State Kinetics ◽

Leaving Group ◽

Transition State Barrier

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13C, 15N, and nonexchangeable 2H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly (kchemlight/kchemheavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster (kchemlight/kchemheavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchemlight/kchemheavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts.

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Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609032114 ◽

2016 ◽

Vol 113 (52) ◽

pp. E8369-E8378 ◽

Cited By ~ 18

Author(s):

Joshua A. Linscott ◽

Kanishk Kapilashrami ◽

Zhen Wang ◽

Chamara Senevirathne ◽

Ian R. Bothwell ◽

...

Keyword(s):

Transition State ◽

Cell Cycle Progression ◽

Cancer Metastasis ◽

Isotope Effects ◽

Kinetic Isotope Effects ◽

Substrate Complex ◽

Flight Mass Spectrometry ◽

Kinetic Isotope ◽

Steady State Kinetics ◽

Lysine Methyltransferase

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)–PKMT–substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactorS-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analogSe-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

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Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704786114 ◽

2017 ◽

Vol 114 (25) ◽

pp. 6456-6461 ◽

Cited By ~ 16

Author(s):

Rajesh K. Harijan ◽

Ioanna Zoi ◽

Dimitri Antoniou ◽

Steven D. Schwartz ◽

Vern L. Schramm

Keyword(s):

Steady State ◽

Transition State ◽

Isotope Effect ◽

Purine Nucleoside Phosphorylase ◽

Isotope Effects ◽

Catalytic Site ◽

Purine Nucleoside ◽

Barrier Crossing ◽

Nucleoside Phosphorylase ◽

Inverse Effect

Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchemlight/kchemheavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kchemlight/kchemheavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre–steady-state chemistry was slowed 32-fold in F159Y PNP. Pre–steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.

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