Ioanna Zoi

Modulating Enzyme Catalysis through Mutations Designed to Alter Rapid Protein Dynamics.

The relevance of sub-picosecond protein motions to the catalytic event remains a topic of debate. Heavy enzymes (isotopically substituted) provide an experimental tool for bond-vibrational links to enzyme catalysis. A recent transition path sampling study with heavy purine nucleoside phosphorylase (PNP) characterized the experimentally observed mass-dependent slowing of barrier crossing (Antoniou, D.; Ge, X.; Schramm, V. L.; Schwartz, S. D. J. Phys. Chem. Lett. 2012, 3, 3538). Here we computationally identify second-sphere amino acid residues predicted to influence the freedom of the catalytic site vibrational modes linked to heavy enzyme effects in PNP. We mutated heavy and light PNPs to increase the catalytic site vibrational freedom. Enzymatic barrier-crossing rates were converted from mass-dependent to mass-independent as a result of the mutations. The mutagenic uncoupling of femtosecond motions between catalytic site groups and reactants decreased transition state barrier crossing by 2 orders of magnitude, an indication of the femtosecond dynamic contributions to catalysis.

Azepinone-Containing Tetrapeptide Analogues of Melanotropin Lead to Selective hMC4R Agonists and hMC5R Antagonist

To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted into the α-MSH pharmacophore, His(6)-Phe(7)-Arg(8)-Trp(9)-domain. Replacement of His(6) by an aminoindoloazepinone (Aia) or aminobenzazepinone (Aba) moiety led to hMC4R and hMC5R selective agonist and antagonist ligands, respectively (tetrapeptides 1 to 3 and 4, respectively). In peptides 1 to 3 and depending on the para-substituent of the d-Phe residue in position 2, the activity goes from allosteric partial agonism (1, R = H) to allosteric full agonism (2, R = F) and finally allosteric partial agonism (3, R = Br).

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

Significance Protein design from first principles is developing rapidly for structural elements, binding domains, and protein–protein interactions. Design of structural elements to generate predictable changes in the fundamental properties of enzymatic catalysis remains challenging, requiring input from protein dynamics and the quantum chemical effects of transition state formation and barrier crossing. Human purine nucleoside phosphorylase (PNP) has a well-understood mechanism of catalysis, which includes rapid protein dynamics. PNP was used in a design program to alter the catalytic-site response to heavy-atom substitution in the enzyme protein. Native PNP exhibits slowed chemistry when made heavy with 2H, 13C, and 15N. We succeeded in designing a second-sphere mutation with improved promoting vibrations to catalyze faster chemistry in response to heavy PNP. 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 (kchem light/kchem 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 heavy-enzyme isotope effect (kchem light/kchem heavy < 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.

Enzyme Homologues Have Distinct Reaction Paths through Their Transition States

Recent studies of the bacterial enzymes EcMTAN and VcMTAN showed that they have different binding affinities for the same transition state analogue. This was surprising given the similarity of their active sites. We performed transition path sampling simulations of both enzymes to reveal the atomic details of the catalytic chemical step, which may be the key for explaining the inhibitor affinity differences. Even though all experimental data would suggest the two enzymes are almost identical, subtle dynamic differences manifest in differences of reaction coordinate, transition state structure, and eventually significant differences in inhibitor binding. Unlike EcMTAN, VcMTAN has multiple distinct transition states, which is an indication that multiple sets of coordinated protein motions can reach a transition state. Reaction coordinate information is only accessible from transition path sampling approaches, since all experimental approaches report averages. Detailed knowledge could have a significant impact on pharmaceutical design.

Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

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

Significance Enzymes achieve catalytic efficiency by optimizing contacts between reactants and catalytic site amino acids. The transition state forms rarely, with a lifetime of a few femtoseconds. Femtosecond motions required for transition state formation are investigated with heavy enzymes containing 2H, 13C, and 15N amino acids to alter bond vibrational modes. Asparagine is a critical amino acid at the catalytic site of human purine nucleoside phosphorylase (PNP). PNP with heavy asparagine, or with all heavy amino acids except asparagine, yields PNPs more efficient at forming the transition state. Computational chemistry reveals that essential catalytic site contacts become more frequently optimized in the labeled enzymes than in the normal enzyme. Heavy enzymes provide unprecedented detail for understanding enzymatic catalysis. 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 (kchem light/kchem heavy = 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 (kchem light/kchem heavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchem light/kchem heavy = 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.

Electric Fields and Fast Protein Dynamics in Enzymes

In recent years, there has been much discussion regarding the origin of enzymatic catalysis and whether including protein dynamics is necessary for understanding catalytic enhancement. An important contribution in this debate was made with the application of the vibrational Stark effect spectroscopy to measure electric fields in the active site. This provided a window on electric fields at the transition state in enzymatic reactions. We performed computational studies on two enzymes where we have shown that fast dynamics is part of the reaction mechanism and calculated the electric field near the bond-breaking event. We found that the fast motions that we had identified lead to an increase of the electric field, thus preparing an enzymatic configuration that is electrostatically favorable for the catalytic chemical step. We also studied the enzyme that has been the subject of Stark spectroscopy, ketosteroid isomerase, and found electric fields of a similar magnitude to the two previous examples.

Incorporating Fast Protein Dynamics into Enzyme Design: A Proposed Mutant Aromatic Amine Dehydrogenase

In recent years, there has been encouraging progress in the engineering of enzymes that are designed to catalyze reactions not accelerated by natural enzymes. We tested the possibility of reengineering an existing enzyme by introducing a fast protein motion that couples to the reaction. Aromatic amine dehydrogenase is a system that has been shown to use a fast substrate motion as part of the reaction mechanism. We identified a mutation that preserves this fast motion but also introduces a favorable fast motion near the active site that did not exist in the native enzyme. Transition path sampling was used for the analysis of the atomic details of the mechanism.