31 - Kinetic Characterization of Transient Free Radical Intermediates in Reaction of Lysine 2,3-Aminomutase by EPR Lineshape Analysis

Organic radicals are increasingly recognized as intermediates in complex enzymatic reactions.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 Radicals have been detected in the steady states of most of the adenosylcobalamin-dependent reactions.2, 3, 4, 5, 6, 7,^9,10 Although in most cases the structures of these radicals have not been fully established, the thiyl-protein radical in B₁₂-dependent ribonucleotide reductase has been characterized.^9,10 Radical intermediates in monooxygenase reactions such as those of cytochrome P450s and methane monooxygenase have been postulated. However, the assignment of radical mechanisms is controversial in these cases because of the indirect nature of the evidence for the participation of organic radicals.11, 12, 13 Radicals have been observed and characterized in the reactions of a few oxygenases, notably lipoxygenase and prostaglandin synthase.^{14, 15}

Because radicals are high-energy species, they often do not accumulate to spectroscopically detectable concentrations in the steady states of enzymatic reactions. Evidence of their presence is limited to observations of product profiles in the reactions of substrates that generate labile intermediates that can undergo fast, radical-mediated isomerizations to produce rearrangement products. The kinetic competence of such putative radicals cannot be unambiguously established.

The most direct evidence for a radical mechanism is the observation of transient radicals by electron paramagnetic resonance spectroscopy (EPR). In these cases, the question of kinetic competence can be addressed experimentally by rapid-mix freeze-quench EPR spectroscopy.¹⁶ This technique allowed the kinetic competence of the thiyl-protein radical in the B12-dependent ribonucleotide reductase to be proven.^9,10,16

The reaction of lysine 2,3-aminomutase (LAM, EC 5.4.3.2) proceeds by the most completely characterized enzymatic radical mechanism.¹,17, 18, 19, 20, 21, 22, 23, 24, 25, 26 LAM catalyzes the interconversion of L-lysinc and L-β-lysine according to Eq. (1), where the migration of the amino group from C-2 to C-3 is accompanied by the countermigration of hydrogen from the 3-pro-R position of L-lysine to the 2-pro-R position of L-β-lysine.²⁷ This isomerization is typical of adenosylcobalamin-dependent reactions; however, the action of LAM does not require a

B₁₂-coenzyme. Instead, LAM contains a [4Fe–4S] center and is activated by S-adenosyl-L-methionine (SAM). The 5´-deoxyadenosyl moiety of SAM mediates the transfer of hydrogen between C-3 of L-lysine and C-2 of L-β-lysine, just as it does in adenosylcobalamin-dependent reactions.^17,18

LAM is a member of the radical SAM superfamily of proteins, which include within their amino acid sequences the unique iron-sulfide motif CxxxCxxC and a SAM-binding motif.²⁸ Other prominent members of this superfamily are SAM-dependent enzymes in which the 5‘-deoxyadenosyl moiety functions as a hydrogen transfer agent, and they include pyruvate formate-lyase activases, bacterial anaerobic ribonucleotide reductases, biotin synthases, lipoyl synthases, and benzylsuccinate synthase activase.²⁸ The superfamily also includes many proteins of imprecisely known or unknown function.

In the reaction of LAM, SAM must be reversibly cleaved to the 5´-deoxyadenosyl radical in order to mediate hydrogen transfer and initiate free radical chemistry. This cleavage is brought about through a reversible chemical reaction between the [4Fe–4S] center and SAM at the active site.²⁹ The process can be described by Eq. (2), in which the highly reduced [4Fe–4S]⁺ center reacts with SAM to transfer an electron, bind methionine, and generate the 5´-deoxyadenosyl radical.

Se-Adenosyl-L-selenomethionine is a good cofactor in place of SAM, and when it activates LAM, Se-XAFS reveals the direct coordination of Se in selenomethionine to the [4Fe-4S] center.³⁰

On recognizing that SAM mediates hydrogen transfer, the radical mechanism in Fig. 1 could be written for the reaction of LAM.¹⁷ Lysine is bound to LAM as its external aldimine with pyridoxal-5´-phosphate (PLP), and the 5´-deoxyadenosyl radical abstracts a hydrogen atom from the 3-pro-R position of the lysyl aldimine to form 5´-deoxyadenosine and lysyl radical 1. Isomerization proceeds through internal radical addition to the aldimine to form the azacyclopropylcarbinyl radical 2, a quasi-symmetric species, which opens in the forward direction to the,β-lysyl radical 3. Abstraction of a hydrogen atom from the methyl group of 5´-deoxyadenosine

produces the β-lysyl aldimine and regenerates the 5´-deoxyadenosyl radical. Reaction of’ L-lysinc with β-lysyl aldimine in several enzymatic steps releases L-β-lysine and binds L-lysine for another cycle of catalysis.

Of the four organic free radicals in the mechanism, three have been observed by EPR spectroscopy. β-Lysyl radical 3 is the most stable radical in the mechanism when L-lysine is the substrate and SAM is the coenzyme, and this is the only radical observed in the steady state of the reaction.20, 21, 22 When 4-thia-L-lysine is the substrate and SAM is the coenzyme, the most stable radical in the steady state is the 4-thia analog of the lysyl radical 1, and this is the only radical observed by EPR.²⁵,²⁶. When the enzyme is activated by 3´,4´-anhydroadenosyl-L-methionine (anSAM) in place of SAM, the most stable radical with L-lysine as the substrate is the

3´,4´-anhydro-5´-deoxyadenosyl radical, an allylic analog of the 5´-deoxyadenosyl radical, and it is the only radical observed in the steady state.^{31 ,32} These three radicals have been characterized spectroscopically by analyzing the EPR spectra with the radicals labeled with deuterium or carbon-13 at various locations, as aids in focusing on the radical centers and the conformations of the radicals.

The spectroscopic observation and characterization of an organic radical in an enzyme proves the presence of the radical but not that it participates in the mechanism. The question of whether the radical is part of the mechanism or a peripheral species must be addressed. An essential property of any reaction intermediate is that it must be formed and react at a rate that is compatible with the overall rate of the reaction. That is, it must be kinetically competent to be an intermediate.

In enzymatic reactions that proceed by radical mechanisms, a method for examining the kinetic competence of a radical is rapid-mix freeze-quench EPR.¹⁶ In the simplest application of this method, the enzyme is mixed in a fast mixing apparatus with the substrates and cofactors that are required to generate the free radical of interest. The reaction is allowed to proceed for a timed interval and then quenched as the sample is sprayed into a very cold organic solvent, such as liquid isopentane. The time from mixing to freezing may be as short as a few milliseconds. The resulting sample contains the reacting species in a chemically arrested state. At the low temperatures of the cryogenic solvent the chemical reactions are very slow in any case, and in the particular case of enzymatic solutions frozen as aqueous droplets translational motion of the molecules is arrested as well, so that the chemical reactions are halted. The snow can then be packed into EPR tubes for analysis by EPR spectroscopy. The snow samples can be collected quantitatively, so that integration of the EPR spectra from packed samples allows the total amount of radical in the sample to be measured. The progress curve of radical formation can be obtained by spray-freezing the enzymatic samples as a function of time and plotting the radical contents of the timed samples against time. Analysis of the progress curve gives the rate constant for radical formation. If this rate constant equals or exceeds the value of k_catfor the reaction, the radical is regarded as kinetically competent.

The rapid mixing machine generally consists of two or three syringes, the barrels of which are connected in variable configurations by tubes, with the plungers being operated by mechanical pressure exerted through one or more push bars. Depending on the complexity of the experiments, several configurations and mixing programs can be arranged. In the simplest experiment like that in the preceding paragraph, only two syringes and one push bar are required. The connection between the syringes and mixer is fixed in volume, but the flexible tubing containing

the mixed sample can be varied in length. The longer the tubing containing the mixed sample, the longer the residence time between the mixer and spray tip, and the reaction time is proportional to the residence time. The reacting time varies with the length of the tube, and the reaction is quenched on being sprayed into the cryogenic solvent. The mechanical operations of commercial machines must be computer controlled to achieve the required accuracy in the timing of mixing and quenching.