Nature of Hydrogen Transfer in Soybean Lipoxygenase 1: Separation of Primary and Secondary Isotope Effects^†

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the α-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in kcat and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76−84) and small secondary (1.1−1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer

Convergent Mechanistic Features between the Structurally Diverse <i>N</i>- and <i>O</i>‑Methyltransferases: Glycine <i>N</i>‑Methyltransferase and Catechol <i>O</i>‑Methyltransferase

Although an enormous and still growing number of biologically diverse methyltransferases have been reported and identified, a comprehensive understanding of the enzymatic methyl transfer mechanism is still lacking. Glycine N-methyltransferase (GNMT), a member of the family that acts on small metabolites as the substrate, catalyzes methyl transfer from S-adenosyl-l-methionine (AdoMet) to glycine to form S-adenosyl-l-homocysteine and sarcosine. We report primary carbon (12C/14C) and secondary (1H3/3H3) kinetic isotope effects at the transferred methyl group, together with 1H3/3H3 binding isotope effects for wild-type GNMT and a series of Tyr21 mutants. The data implicate a compaction effect in the methyl transfer step that is conferred by the protein structure. Furthermore, a remarkable similarity of properties is observed between GNMT and catechol O-methyltransferase, despite significant differences between these enzymes with regard to their active site structures and catalyzed reactions. We attribute these results to a catalytically relevant reduction in the methyl donor–acceptor distance that is dependent on a tyrosine side chain positioned behind the methyl-bearing sulfur of AdoMet

Investigating Inner-Sphere Reorganization via Secondary Kinetic Isotope Effects in the C−H Cleavage Reaction Catalyzed by Soybean Lipoxygenase: Tunneling in the Substrate Backbone as Well as the Transferred Hydrogen

This work describes the application of NMR to the measurement of secondary deuterium (2° 2H) and carbon-13 (13C) kinetic isotope effects (KIEs) at positions 9−13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° 2H and 13C KIEs on kcat/Km directly for 11,11-d2-LA, whereas the values for the 2° 2H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° 2H and 13C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C−H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° 2H KIEs

Enzymatic Methyl Transfer: Role of an Active Site Residue in Generating Active Site Compaction That Correlates with Catalytic Efficiency

Human catechol-<i>O</i>-methyltransferase (COMT) catalyzes a methyl transfer from <i>S</i>-adenosylmethionine (AdoMet) to dopamine. Site-specific mutants at three positions (Tyr68, Trp38, and Val108) have been characterized with regard to product distribution, catalytic efficiency, and secondary kinetic isotope effects. The series of mutations at Tyr68 within wild-type protein and the common polymorphic variant (Val108Met) yields a linear correlation between the catalytic efficiency and the size of the secondary kinetic isotope effect. We conclude that active site compaction in COMT is modulated by a proximal side chain residing behind the sulfur-bearing methyl group of AdoMet. These findings are discussed in the context of the active site compression that has been postulated to accompany enzyme-supported hydrogen tunneling

Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase

Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (<i>k</i>_cat/<i>K</i>_m) and compared to the unimolecular rate constant, <i>k</i>_cat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (<i>k</i>_cat/<i>K</i>_m) are all reduced from ^D<i>k</i>_cat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is <i>orthogonal</i> to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis

Comparison of Rates and Kinetic Isotope Effects Using PEG-Modified Variants and Glycoforms of Glucose Oxidase: The Relationship of Modification of the Protein Envelope to C−H Activation and Tunneling^†

An earlier investigation of the temperature dependencies of rates and kinetic isotope effects (KIEs) in glucose oxidase (GO) used variants that differed in the extent of glycosylation at the surface of the protein. Kohen et al. [Kohen, A., Jonsson, T., and Klinman, J. P. (1997) Biochemistry 36, 2603−2611] presented evidence that the KIE on the Arrhenius prefactor varied as a function of protein modification, concluding that the degree of hydrogen tunneling at the active site was dependent on changes in mass at the surface. We now examine GO proteins containing polyethylene glycol (PEG) at their surface and a more extensively glycosylated form of GO, to distinguish simple mass effects from other sources of altered catalytic behavior. One PEG variant was created by modifying deglycosylated GO with short PEG chains (average of 350 Da each), while another contained a smaller number of long PEG chains (average of 5000 Da each). The light (146 kDa) and heavy (211 kDa) PEG variants and the hyperglycosylated variant display isotope effects on the Arrhenius prefactor that are similar (AD/AT = 0.55−0.62), while the unperturbed wild-type GO (WT-GO) is found to have an AD/AT that is reassessed as being close to unity. It appears that any modification of the protein surface away from that of the wild type gives rise to altered behavior for hydrogen transfer in the active site. We have also compared the effect of enthalpies of activation on both kcat/KM and kcat for the variants, introducing a new method to extract the kcat/KM rate constant and enthalpy of activation for the tritiated substrate from competitive KIE experiments. We find similar trends in ΔH⧧ for both competitive and noncompetitive parameters and a smaller trend in kcat than reported earlier. Correlations are observed between AD/AT and both the enthalpies of activation and the thermal melt temperatures (TM) of the GO isoforms. In addition to the present study, there are now a number of examples where a perturbation of enzyme structure away from that of the wild type causes the observed KIE to become more temperature-dependent. The implications of these findings are discussed in the context of hydrogen tunneling and the relationship of protein structure and dynamics to this process

Experimental Evidence for Hydrogen Tunneling when the Isotopic Arrhenius Prefactor (<i>A</i>_H/<i>A</i>_D) is Unity

Experimental Evidence for Hydrogen Tunneling when the Isotopic Arrhenius Prefactor (AH/AD) is Unit

Hydrogen Tunneling in a Prokaryotic Lipoxygenase

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ∼25% smaller catalytic domain, and an increase in <i>E</i>_a of ∼11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an <i>E</i>_a similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences. These findings, which extend the phenomenon of hydrogen tunneling to a prokaryotic LOX, are discussed in the context of a role for protein size and/or flexibility in enzymatic hydrogen tunneling

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosyl­methionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein–protein and protein–peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosyl­methionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes