Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

Javier Suarez, Vern L. Schramm

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (<sup>13</sup>C, <sup>15</sup>N, and <sup>2</sup>H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [<sup>2</sup>H]PNP and [<sup>13</sup>C, <sup>15</sup>N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [<sup>15</sup>N, <sup>2</sup>H]His<inf>8</inf>-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [<sup>15</sup>N, <sup>2</sup>H]His<inf>8</inf>-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transitionstate formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

Original languageEnglish (US)
Pages (from-to)11247-11251
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number36
DOIs
StatePublished - Sep 8 2015

Fingerprint

Purine-Nucleoside Phosphorylase
Isotopes
Catalytic Domain
Amino Acids
Deuterium
Carbon
Enzymes
Proteins

Keywords

  • Born-Oppenheimer enzymes
  • Femtosecond dynamics
  • Heavy enzymes
  • Pre-steady-state chemistry
  • Transition state coupling

ASJC Scopus subject areas

  • General

Cite this

@article{e6f0a99d86104dec9e9322b6e71ab9ff,
title = "Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase",
abstract = "Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transitionstate formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.",
keywords = "Born-Oppenheimer enzymes, Femtosecond dynamics, Heavy enzymes, Pre-steady-state chemistry, Transition state coupling",
author = "Javier Suarez and Schramm, {Vern L.}",
year = "2015",
month = "9",
day = "8",
doi = "10.1073/pnas.1513956112",
language = "English (US)",
volume = "112",
pages = "11247--11251",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "36",

}

TY - JOUR

T1 - Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

AU - Suarez, Javier

AU - Schramm, Vern L.

PY - 2015/9/8

Y1 - 2015/9/8

N2 - Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transitionstate formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

AB - Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transitionstate formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

KW - Born-Oppenheimer enzymes

KW - Femtosecond dynamics

KW - Heavy enzymes

KW - Pre-steady-state chemistry

KW - Transition state coupling

UR - http://www.scopus.com/inward/record.url?scp=84941068055&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84941068055&partnerID=8YFLogxK

U2 - 10.1073/pnas.1513956112

DO - 10.1073/pnas.1513956112

M3 - Article

C2 - 26305965

AN - SCOPUS:84941068055

VL - 112

SP - 11247

EP - 11251

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 36

ER -