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

Abstract

Heavy-enzyme isotope effects ( 15 N-, 13 C-, and 2 H-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 heavyenzyme isotope effect (k chem light /k chem heavy > 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 heavyenzyme isotope effect (k chem light /k chem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steadystate 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.heavy enzyme | transition path sampling | purine nucleoside phosphorylase | enzyme design | femtosecond dynamics D ynamic motions essential for enzyme catalysis occur on timescales from milliseconds for conformational changes to femtosecond bond vibrations associated with chemistry at catalytic sites. The millisecond motions are linked to structural changes during substrate binding (1) and product release, whereas the femtosecond motions are involved in transition state (TS) formation. Alterations of the femtosecond dynamics by isotope substitution in enzymes influence the probability of TS barrier crossing when protein femtosecond motions are coupled to chemistry at catalytic sites (2, 3).The femtosecond dynamical effects in heavy purine nucleoside phosphorylase (PNP) on catalysis have been observed experimentally and have been explained by computational transition path sampling (TPS) (4, 5). TPS can provide insight into the atomic details of chemical reactions without prior knowledge of the reaction coordinate (6-8).Human PNP is a homotrimer that catalyzes the reversible phosphorolysis of 6-oxypurine nucleosides and 6-oxypurine-2′-deoxynucleosides to generate the corresponding purine bases and α-D-ribose (or 2-deoxy-α-D-ribose) 1-phosphates (Fig.

Heavy Enzymes and the Rational Redesign of Protein Catalysts

“…Strategies previously employed in the literature by various groups include isotopically labelling the entire enzyme or labelling either of single amino acid residues or of particular segments, such as mobile loops (Figure ) . The effects of protein isotope labelling on transition states have been characterised in different enzymes including purine nucleotide phosphorylase (PNP), HIV protease (HIV‐1 PR), alanine racemase, dihydrofolate reductase (DHFR), pentaerythritol tetranitrate reductase (PETNR), formate dehydrogenase (FDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase . In most of these cases, isotope labelling reduced the rate of the chemical step ––.…”

“…Theoreticians have proposed that introducing a new “promoting motion” would improve the activity of aromatic amine dehydrogenase but this has never been tested experimentally . Similarly, work on PNP has shown that the efficiency of barrier crossing in a heavy enzyme can be modified by mutations that enhance promoting vibrations . Experimentally, this resulted in the inversion of the enzyme KIE from a normal KIE of 1.31 to an inverse KIE of 0.75 .…”

“…Similarly, work on PNP has shown that the efficiency of barrier crossing in a heavy enzyme can be modified by mutations that enhance promoting vibrations . Experimentally, this resulted in the inversion of the enzyme KIE from a normal KIE of 1.31 to an inverse KIE of 0.75 . However, the mutant enzyme was less catalytically efficient than the wild type.…”

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

“…Few reports have indicated that heavy enzymes can have faster chemistry than the light counterpart. With PNP, TPS explored the possibility that a mass-altered catalytic site mutant protein could be designed to exhibit increased chemistry rates relative to its unlabeled counterpart at the catalytic site (19). The analysis indicated that altering phenylalanine 159 to tyrosine (F159Y) would create an enzyme where the femtosecond dynamics of the heavy enzyme were more likely to find the transition state than the same enzyme in its natural isotopic form.…”

“…This protein design element from TPS was confirmed by demonstrating a k chem light / k chem heavy of 0.75. However, the F159Y mutation in PNP also caused less efficient catalytic site chemistry, slowing it by a factor of 32 (19). An inverse heavy-enzyme isotope effect was also Significance Enzymes achieve catalytic efficiency by optimizing contacts between reactants and catalytic site amino acids.…”