pH-Independent Heat Capacity Changes during Phosphorolysis

doi.org/10.26434/chemrxiv.14135840.v1

pH-Independent Heat Capacity Changes during PhosphorolysisCatalyzed by the Pyrimidine Nucleoside Phosphorylase from GeobacillusthermoglucosidasiusFelix Kaspar, Darian S. Wolff, Peter Neubauer, Anke Kurreck, Vickery Arcus

Submitted date: 01/03/2021 • Posted date: 02/03/2021Licence: CC BY 4.0Citation information: Kaspar, Felix; Wolff, Darian S.; Neubauer, Peter; Kurreck, Anke; Arcus, Vickery (2021):pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine NucleosidePhosphorylase from Geobacillus thermoglucosidasius. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.14135840.v1

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation,indicative of a change in activation heat capacity. However, pH and (de)protonation effects on thisphenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilicpyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensionalworking space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Ouranalysis revealed the presence of a measurable activation heat capacity change in this reaction system, whichshowed no significant dependence on medium pH or substrate charge. Our results further describe theremarkable effects of a single halide substitution which has a minor influence on the heat capacity change butconveys a significant kinetic effect by lowering the activation enthalpy, causing a >10-fold rate increase.Collectively, our results present an important piece in the understanding of enzymatic systems acrossmultidimensional working spaces where the choice of reaction condition can affect rate, affinity andthermodynamic phenomena independently of one another.

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1

pH-Independent Heat Capacity Changes during Phosphorolysis

Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus

thermoglucosidasius

Felix Kaspar,1,2* Darian S. Wolff,1 Peter Neubauer,1 Anke Kurreck,1,2 Vickery L. Arcus 3

1 Chair of Bioprocess Engineering, Institute of Biotechnology, Faculty III Process Sciences, Technische Universität Berlin, Straße des 17. Juni 135, D-10623, Berlin, Germany.

2 BioNukleo GmbH, Ackerstraße 76, D-13355 Berlin, Germany. 3 Te Aka Mātuatua - School of Science, Te Whare Wānanga o Waikato - University of Waikato,

Hamilton 3240, New Zealand. Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, pH and (de)protonation effects on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus

thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change Δ��‡ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution which has a minor influence on Δ��‡ but conveys a significant kinetic effect by lowering the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect rate, affinity and thermodynamic phenomena independently of one another. Temperature is a central variable for the rates of chemical reactions. Most first-order reactions follow an exponential rate-temperature relationship as described by the Eyring equation.[1,2]

� = ��ℎ �� −Δ�‡ + �Δ�‡�� (1)

Herein, � is the rate constant, � is the reaction temperature, � is the universal gas constant and �� and ℎ are the Boltzmann and Planck constants, respectively. Δ�‡ and Δ�‡ are the enthalpy and entropy change between the reactants and the transition state. The transmission coefficient κ is presumed to be 1 hereafter. Despite this equation holding remarkably well for most chemical transformations, there is a growing consensus that enzyme-catalyzed reactions can deviate from the rate-temperature relationship predicted by the Eyring equation by displaying a curvature in their Eyring plots.[3,4] This behavior has been shown to originate from a heat capacity change between the enzyme substrate complex and the enzyme transition state complex, caused by pre-organization of the enzyme along the reaction coordinate (i.e. during transition state binding).[5–14] This molecular property has been described by the Macromolecular Rate Theory (MMRT), which extends the Eyring equation by terms accounting for activation heat capacity changes.[5]

� = ��ℎ �� −Δ��‡ − Δ��‡(� − ��)��+ ΔS��‡ + Δ��‡(�� − ��)� �

(2)

Here, Δ��‡ is the change in activation heat capacity and Δ��‡

and ΔS��‡ are the activation enthalpy and entropy,

respectively, at an arbitrary reference temperature ��. Thus, MMRT provides a molecular explanation for the observed curvature in Eyring plots in the absence of denaturation, which has been demonstrated for a variety of biocatalytic reactions (see Table S1 for a list). Nonetheless, previous characterizations of rate-temperature relationships of various enzymes via Eyring plots have generally been performed at only one pH value (typically pH 7 in phosphate buffer, Table S1), leaving the pH dimension of the reaction space, as well as protonation effects on any of the activation parameters in equations (1) and (2), largely unexplored.

In this context we became interested in the rate-temperature relationships of thermostable nucleoside phosphorylases (NPs)[15] as these enzymes allow an experimental coverage of both a broad pH and temperature window. NPs perform the reversible phosphorolysis of nucleosides (Figure 1A) and are key catabolic enzymes in all kingdoms of life.[16] Owing to their important role in various diseases,[17–22] their catalytic properties have received considerable attention. Furthermore, NPs are used in synthetic chemistry for the preparation of pentose-1-phosphates and nucleoside analogs[23–28] and have been employed as analytical tools.[29] While purine NPs have been researched intensively,[30–36] including their rate-temperature relationships,[10,37] comparably little is known about rate-temperature relationships of reactions catalyzed by the structurally distinct pyrimidine NPs.[38] Since these enzymes typically operate across a wide pH window with similar rate constants[39,40] and convert electronically diverse substrates,[38,39] we hypothesized that they would present a convenient model system to interrogate pH and deprotonation effects on the Eyring plots of nucleoside phosphorolysis, as an example of a simple nucleophilic

Kaspar et al. 2021

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substitution. Thus, we questioned if there exists an activation heat capacity change during the phosphorolysis reactions catalyzed by thermostable pyrimidine NPs and, if so, whether this effect shows any pH- and/or protonation-dependence across the broad working space of these enzymes. Herein, we report a kinetic characterization of the thermophilic pyrimidine NP from Geobacillus

thermoglucosidasius (GtPyNP) across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values (thymidine, 1a, pKa ≈ 10.0 and 2’-deoxy-5-fluorouridine, 1b, pKa ≈ 7.3,[41] Figure 1A).

Figure 1. Phosphorolysis of the nucleosides 1a and 1b catalyzed by GtPyNP (A) and its rate-temperature relationship at pH 7 (B). Both pKa values listed in A represent approximate values at 25 °C and are subject to slight changes at higher temperatures. Data in B were acquired via sampling and discontinuous monitoring of reactions with a high-throughput assay[42,43] and fitted to the Eyring (1) and MMRT (2) equations, as described in the Supplementary Information. Please see the externally hosted Supporting Information for raw data and calculations.[44]

First, we assayed GtPyNP for phosphorolytic conversion

of 1a and 1b at pH 7, employing deconvolution of UV absorption spectra for reaction monitoring.[42,43] To this end, several preliminary experiments were essential, as we aimed to acquire activity data unaffected by denaturation effects (TM = 77.5 °C, Figure S1), which required the selection of experimental conditions enabling sufficient stability of the enzyme (please see the Supplementary Information for details). Employing saturating substrate concentrations and a temperature window of 30−65 °C, the methylated nucleoside 1a was converted with observed rate constants

�� of 2.1−30.3 s-1, showing a clear temperature-dependence (Figure 1B). Both the Eyring and the MMRT equation provided reasonably good fits of the experimental data (R2 = 0.84 and 0.86). However, the Eyring fit showed a systematic deviation from the data by overestimating at the extremes of the temperature and underestimating in the middle of the dataset, while also suggesting a considerable enthalpy-entropy trade-off (Δ�‡ = 49.9 kJ mol-1 and Δ�‡ = -71.8 J mol-1 K-1). In contrast, the MMRT fit provided a more realistic description of the dataset by reflecting the experimental data more evenly throughout the full temperature range, as supported by the Akaike information criterion (AIC, Table S3).[45] This fit yielded a heat capacity change Δ��‡ of -1.9 ± 0.7 kJ mol-1 K-1, manifesting itself in a moderate but significant curvature in the graph. A similar observation was made for the fluorinated nucleoside 1b, whose phosphorolysis showed a Δ��‡ of -1.2 ± 0.6 kJ mol-1 K-1. This suggests a comparable binding of the transition states of both substrates, which is likely rooted in the nearly equal steric demand of these substrates as well as their identical electronic interactions with the H-bonding residues in the GtPyNP active site under these conditions. In contrast to 1a, the fluorinated 1b was converted with much higher rate constants of 69.7−406.6 s-1. Interestingly, this increase of �� originated from a significantly lower activation enthalpy (Δ� !"#‡ = 92.2 kJ mol-1 for 1a and 62.5 kJ mol-1 for 1b), which outweighed the lower entropic contribution (Table S3) and most likely reflects a weaker glycosidic bond caused by electron withdrawal through the fluorine substituent. In line with the lower activation enthalpy observed for 1b, its phosphorolysis showed a shallower temperature-dependence, reflected by a rate increase of factor 4.9 between 30 and 65 °C, compared to a factor of 8.6 for 1a.

Collectively, these results provide several insights. First, despite disagreeing literature,[46] this untangling of activity and denaturation effects revealed that GtPyNP possesses no classical optimum temperature.[5] Previous assignments of an optimum temperature of this enzyme[46] can likely be ascribed to enzyme denaturation since its theoretical optimum temperature is approximately equal to its melting temperature (TM = 77.5 °C, theoretical Topt = 75 °C for 1a or 79 °C for 1b, Figures S1 and S6). Secondly, the data presented above lend further evidence to the notion that binding of the substrate and binding of the transition state are unrelated phenomena,[47] as enzymes primarily discriminate between substrates on the transition state level rather than via selective binding of the ground states of substrates. This is evident here in the observed difference of the affinities for the two substrates (KM = 0.6 mM for 1a and 3.1 mM for 1b) and the similarity of Δ��‡ (-1.9 and -1.2 kJ mol-1 K-1). Thirdly, considering previous work indicating that the overall phosphorolysis of 1a and 1b proceeds with almost the same equilibrium state thermodynamics,[39] these results indicate that electron withdrawal by the fluorine substituent primarily has a kinetic effect in this reaction system and almost no thermodynamic one. This is reflected by the lower Δ�‡ for 1b compared to 1a contrasting their almost identical equilibrium constants (Keq = 0.15 for 1a and 0.12 for 1b) and apparent net thermodynamic parameters Δ� and Δ� (Scheme S2).[39]

Kaspar et al. 2021

3

Next, we extended this kinetic characterization to the entire pH range accessible with GtPyNP to examine the impact of reaction pH and substrate deprotonation on the kinetic behavior of this system. We hypothesized that the medium pH might influence the surface charge of the protein and, consequently, its molecular dynamics, which might be reflected by the heat capacity changes during catalysis. Furthermore, 1b (pKa ≈ 7.3)[41] becomes fully deprotonated at higher pH values and we expected to see pH effects on its activation thermodynamics. To probe these hypotheses, we obtained Eyring plots for the phosphorolysis of 1a and 1b from pH 7−10 in steps of 0.5 pH units (Figure S4). In all but one condition, the AIC supported the MMRT model (2) as the better fit, substantiating the presence of a negative Δ��‡ (Table S3, Figure 2). Unexpectedly, the reaction pH generally had no significant impact on the phosphorolysis kinetics with 1a as �� and all fit parameters remained largely unchanged across the working space (Figure 2A). Despite its deprotonation in the upper portion of the working space, a similar situation existed for 1b, albeit with a much lower and error-prone Δ�‡ (Figure 2B). Although deprotonation severely affected GtPyNP’s affinity for this substrate (KM >10 mM at pH 9 compared to 3.1 mM at pH 7, Figure S3D), it had no significant effect on the thermodynamic activation parameters, suggesting that transition state formation and binding are unaffected by the net charge of this substrate. Most likely, deprotonation of the substrate occurs per se in the active site of the enzyme where an anionic species would be well stabilized by positively charged residues (Scheme S1 and e.g. pdb ID 1uou or 2wk6). As such, considering this mode of substrate complexation in pyrimidine NP active sites, it is reasonable to assume that even a partial dianion present during such a transformation can be accommodated (Scheme S1). Overall, the results of these experiments indicate that GtPyNP-catalyzed phosphorolysis is strikingly robust and shows little sensitivity to pH shifts or the protonation state of its nucleoside substrate, despite charge delocalization being a primary mechanism of rate acceleration in pyrimidine NPs.[48] The fact that we obtained very similar Eyring plots over a broad pH range further indicates that the surface charge of the protein insignificantly contributed to the molecular dynamics and, in extension, activation heat capacity changes along the reaction coordinate. Nonetheless, it remains to be demonstrated if this behavior observed for the dimeric GtPyNP with a solvent-shielded active site also translates to highly multimeric enzymes or those with solvent-exposed active sites.

In conclusion, our kinetic characterization of GtPyNP revealed the presence of a measurable activation heat capacity change Δ��‡, which showed no significant dependence on medium pH or substrate charge. Experiments across a wide working space uncovered the remarkable effects of a single halide substitution which has a minor influence on Δ��‡ but conveys a significant kinetic effect by lowering the activation enthalpy, causing a >10-fold rate increase. Therefore, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect rate, affinity and thermodynamic phenomena independently of one another.

Figure 2. Activation thermodynamics of the GtPyNP-catalyzed phosphorolysis of 1a (A) and 1b (B) across the accessible working space. For clarity, errors (shaded bars) are only shown in one direction. Please see Table S3 for tabulated data as well as the externally hosted Supporting Information for raw data and calculations.[44]

Acknowledgements

The authors thank Kerstin Heinecke (TU Berlin) for proofreading and critical comments. Conflict of Interest

A. K. is CEO of the biotech company BioNukleo GmbH. F. K. is a scientist at BioNukleo GmbH and P. N. is a member of the advisory board. These affiliations constitute no conflict of interest with the results presented and discussed in this report. Author Information

Corresponding Author

Felix Kaspar, [email protected], orcid.org/0000-0001-6391-043X Other Authors

Darian S. Wolff, orcid.org/0000-0002-9266-3017 Dr. Peter Neubauer, orcid.org/0000-0002-1214-9713 Dr. Anke Kurreck, orcid.org/0000-0001-6919-725X Dr. Vickery L. Arcus, orcid.org/0000-0001-5082-2414

Kaspar et al. 2021

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download fileview on ChemRxivKaspar_2021_GtPyNP_MMRT.pdf (2.56 MiB)



1

Supporting Information

Felix Kaspar,1,2 Darian S. Wolff,1 Peter Neubauer,1 Anke Kurreck,1,2 Vickery L. Arcus3

1 Chair of Bioprocess Engineering, Institute of Biotechnology, Faculty III Process Sciences, Technische

Universität Berlin, Straße des 17. Juni 135, D-10623, Berlin, Germany

2 BioNukleo GmbH, Ackerstraße 76, D-13355, Berlin, Germany

3 Te Aka Mātuatua - School of Science, Te Whare Wānanga o Waikato - University of Waikato,

Hamilton 3240, New Zealand

Author contributions 1

Data availability 1

General remarks 2

Experimental 2

Selection of reaction conditions 4

Supplementary items 5

Table S1 5

Melting point of GtPyNP (Figure S1) 6

Half-life of GtPyNP (Figure S2 and Table S2) 6

Michaelis-Menten kinetics (Figure S3) 8

Eyring plots (Figure S4 and Table S3) 10

Buffer system (Figure S5) 13

Theoretical optimum temperatures (Figure S6) 13

Mechanism and energy profile (Schemes S1 and S2) 14

Supplementary references 15

Author contributions (with definitions as recommended by Brand et al.[1])

Conceptualization, F.K.; Data curation, F.K. and D.S.W.; Formal analysis, F.K. and D.S.W.; Funding

acquisition, P.N. and A.K.; Investigation, F.K. and D.S.W.; Methodology, F.K.; Project administration,

F.K., A.K. and V.L.A.; Resources, P.N. and A.K.; Software, - ; Supervision, P.N., A.K. and V.L.A.; Validation,

- ; Visualization, F.K.; Writing—original draft, F.K.; Writing—review & editing, F.K., D.S.W., P.N., A.K.

and V.L.A.

Data availability

All data depicted visually in the items in the main text (Figures 1 and 2) as well as in the Supplementary

Information (Figures S1−S6 and Tables S2 and S3, see below) are available as tabulated data from the

externally hosted Supporting Information at zenodo.org.[2]

Kaspar et al. Supporting Information

2

General remarks

All chemicals used in this study were of analytical grade or higher and purchased from Sigma Aldrich

(Steinheim, Germany), Carbosynth (Berkshire, UK), Carl Roth (Karlsruhe, Germany), TCI Deutschland

(Eschborn, Germany) or VWR (Darmstadt, Germany) and used without prior purification. Water

deionized to 18.2 MΩ∙cm with a Werner water purification system was used for the preparation of all

enzymatic reactions as well as purification and storage buffers. For the preparation of NaOH solutions

for quenching, deionized water was used.

Experimental

Protein expression was performed as described previously.[3,4] Briefly, GtPyNP was heterologously

expressed in E. coli as a His6-tagged protein through IPTG-induced overexpression. Purification was

achieved through cell disruption, heat treatment of the crude extract (60 °C for 30 min) and Ni-NTA

affinity chromatography. The target protein was eluted with buffer containing 250 mM imidazole,

50 mM sodium phosphate, 300 mM NaCl (pH 8) and stored as stock solutions at −20 °C in 50% (v/v)

glycerol in 2 mM potassium phosphate buffer (pH 7) at a stock concentration of 1.2 g L-1 (calculated

with 1 AU cm-1 at 280 nm being equal to a protein concentration of 1 g L-1). Under these storage

conditions, no decay of activity could be detected over the course of more than 1 year. GtPyNP has an

extinction coefficient of 21,890 cm-1 M-1 as predicted by Protparam,[5] thus the stock solution of 1.2 g L-1

had a concentration of 54.8 µM.

Enzymatic reactions were performed in 1.5 mL Eppendorf tubes and prepared from stock solutions of

nucleoside, enzyme and phosphate, hydrazine, borate (PHB) buffer. Typical reaction volumes were 200

or 500 µL, depending on the experiment and substrate concentration. Reactions were preheated to

the respective temperature for 30 s prior to the reaction and initiated by the addition of suitably

diluted enzyme stock solution (predilution in PHB buffer with the respective pH value). Samples were

withdrawn at timely intervals, typically after 20, 40 and 60 s after reaction initiation or as detailed in

the metadata files freely available online.[6] Unless stated otherwise, all reactions were performed in

triplicate. The buffer system employed in this study was composed of equimolar amounts of

phosphate, hydrazine and borate (Figure S5). To this end, sodium phosphate, hydrazine monohydrate

and sodium borate were dissolved in water (400 mM each) and the resulting solution (ca. pH 7) was

aliquoted for pH adjustment. This mother buffer was adjusted to pH values of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5

and 10.0 using 1 M HCl and 5 M NaOH. These aliquots were then equated for ionic strength

(considering all charged species) with 5 M NaCl and diluted to 200 mM buffer components to give

buffer stocks with an ionic strength of 1 M. Since our kinetic experiments were all performed at 50 mM

buffer strength (except the KM determination for phosphate), enzymatic reactions were run at an ionic


3

strength of 250 mM. GtPyNP is remarkably insensitive to ionic strength and tolerates such high ion

concentrations well, which we confirmed in initial experiments (data not shown).

Reaction monitoring was achieved via spectral unmixing. From live reactions, samples were withdrawn

and quenched in aqueous NaOH as described previously.[4,7] For the substrates 1a, samples were

quenched in 100 mM NaOH and for 1b in 200 mM NaOH, as detailed in our previous work.[7] Sample

dilution factor was adjusted to reach final concentrations of 100−150 µM UV-active reaction

components (please note that the exact concentration is not relevant here since spectral unmixing

only takes spectral shape and not absolute intensity into account). Of the diluted alkaline sample,

200 µL were transferred to UV/Vis-transparent 96-well plates (UV star, GreinerBioOne, Kremsmünster,

Austria) for analysis. UV absorption spectra were recorded from 250−350 nm with a BioTek

PowerWave HT platereader and subjected to spectral unmixing using analogously obtained reference

spectra.[6] The reference spectra used in this study are freely available in the externally hosted

Supplementary Information[2] and some of these can, alternatively, be obtained from the

Supplementary Information of our previous publication.[8] The degree of conversion was determined

directly from the spectra fit which considers the UV-active substrate and product in relation to one

another.[4] For activity determination, only sampling points showing 2−12% conversion of the

nucleoside substrate were considered. This lower bound was set due to the inherent inaccuracy of the

UV-based method employed (roughly ±0.3 percentage points, due to the inherent error in spectral

acquisition, as described in the original publication)[4] and the upper bound was applied as

recommended by Cornish-Bowden[9] for equilibrium reactions. All collected datapoints outside this

window were not included for calculation of activity and marked accordingly in the Supplementary

Information.[2] Datapoints that displayed baseline shifts or other spectral anomalies were also excluded

from consideration. Background correction was performed as described recently.[7] Experimental

spectra were fitted either across the entire spectrum or over one of the information-rich shoulder

regions of pyrimidine nucleosides/nucleobases, as appropriate for the analysis. All background

corrections, fitting wavelengths and the corresponding datafiles are detailed in the metadata files in

the externally hosted Supplementary Information.[2,6]

Rate constants of phosphorolysis were determined by linear approximation of the conversion over

time with a forced intercept at the origin. All raw data and the datapoints considered for calculation

are freely available online with outliers and excluded datapoints clearly marked.[6] The observed rate

constant �� was obtained by considering the degree of conversion (mol per second) per mol enzyme

applied, using the molar extinction coefficient of GtPyNP of 21,890 cm-1 M-1 as predicted by

Protparam.[5]


4

Selection of reaction conditions

For our kinetic characterization of GtPyNP, several preliminary experiments were essential, as we

aimed to acquire activity data unaffected by denaturation effects, which required the selection of

experimental conditions enabling sufficient stability of the enzyme. Since we sought to obtain Eyring

plots across the entire pH range reliably accessible with GtPyNP (pH 7−10), we employed an inorganic

buffer system containing the temperature-insensitive buffer components phosphate (pKa = 7.2),

hydrazine (pKa = 8.0) and borate (pKa = 9.2). Herein, phosphate acted both as a buffer and as a

substrate present in excess, driving the equilibrium in the phosphorolysis direction (Keq = 0.15 for 1a

and 0.12 for 1b).[10] As borates are known to interfere with substrates containing cis-1,2-diols,[11] we

intentionally opted for the 2’-deoxyribosides 1a and 1b to characterize GtPyNP in this buffer system.

Using 50 mM of each buffer component and solutions equated for ionic strength, GtPyNP displayed

slightly pH-dependent melting points of 77.5−78 °C (Figure S1). The apparent onset of denaturation at

65 °C indicated that this temperature would likely be the upper limit allowing us to obtain kinetic data

unaffected by denaturation or inactivation effects. To confirm this hypothesis, we tested the half-life

of the enzyme at 65 °C and found values of >60 min from pH 7 to 9. Only the high pH regime of pH 9.5

and 10 affected the stability of GtPyNP at this temperature, with half-lives of 52 and 35 min,

respectively (Figure S2 and Table S2), permitting activity assays in the 1 min time domain. Therefore,

we selected 65 °C as the upper bound for the investigated temperature range. Next, we examined the

substrate affinities of GtPyNP to enable a selection of substrate concentrations at or near saturating

levels, to minimize affinity effects on the Eyring plots. At pH 7, GtPyNP showed KM values of 0.6 mM

for 1a and 3.1 mM for 1b with only a minor temperature-dependence (Figure S3A and 3C) as well as a

KM of ca. 1 mM for phosphate (Figure S3E). Based on these data, we selected substrate concentrations

of 2 mM for 1a, 5 mM for 1b and 50 mM for phosphate for the following kinetic experiments to work

near saturating concentrations.


5

Supplementary Items

Table S1. Known enzymes with non-zero Δ��‡

Enzyme Reaction Buffer pH Reference

Glucosidase MalL Carbohydrate hydrolysis Phosphate 7.0 [12]

3-isopropylmatalte

dehydrogenase LeuB Dehydrogenation Phosphate 7.6 [13]

Barnase Nucleotide hydrolysis Phosphate 7.0 [12]

Ketosteroid isomerase Olefin isomerization Phosphate 7.0 [14]

S-Methyl-5'-

thioadenosinphosphorylase

(MTAP)

Nucleoside phosphorolysis Phosphate 7.4 [15]

Acid phosphatase Phosphorylation Acetate 5.0 [16,17]

Alkaline phosphatase Phosphorylation Diethanol-

amine 8.5 [18]

Adenosine deaminase Nucleoside deamination Phosphate 7.4 [16,17]

Aryl-acylamidase Arene hydrolysis Tris 8.6

-Lactamase Lactam hydrolysis Phosphate 7.0 [16,17]

Tyrosine phenol-lyase Amino acid hydrolysis Triethanol-

amine 8.0 [19]

Monoamine oxidase MAO-B Amine oxidation HEPES 7.5 [20]

Glucose dehydrogenase Carbohydrase oxidation HEPES 8.0 [21]

Kemp eliminase Deprotonation Phosphate 7.0 [22]


6

The melting point of GtPyNP in PHB buffer was determined by a thermal shift assay. To this end,

GtPyNP (0.2 g/L) and SYPRO orange (5x) were incubated in 50 mM PHB buffer in a total volume of

25 µL in sealed wells of a PCR plate. The solutions were incubated at 50 °C for 30 s and subsequently

heated in steps of 0.5 °C per 5 s to a final temperature of 95 °C in a Biorad CFX96 Real-Time system. At

each 0.5 °C interval, fluorescence was measured (λex = 470 nm, λem = 570 nm). The melting point TM

was determined as the inflection point of the fluorescence intensity over the temperature (equal to

the extreme of the first derivative of the fluorescence as shown in Figure S1).

Figure S1. Melting point of GtPyNP as a function of pH.

The half-life of GtPyNP at 65 °C was determined by incubation of GtPyNP (4.0 µg mL-1) in 50 mM PHB

buffer at pH 7−10 and 65 °C in a total volume of 220 µL in a PCR tube (please note that the PCR tube

was chosen intentionally for incubation since it ensures homogenous heating of the entire mixture

without any sample cooling or evaporation). The tubes were incubated in a PCR cycler with lid heating

for various timespans at 65 °C (0, 30 and 60 min for pH 7−9 and 0, 10, 20, 30 and 60 min for pH 9.5 and

10). The lid temperature was set to 75 °C to prevent sample condensation at the top of the tubes. At

the indicated timepoints, tubes were removed from the PCR cycler and immediately cooled on ice until

activity determination. To assay for (residual) enzymatic activity, 150 µL of the incubated enzyme

mixture were used to start a reaction consisting of 1 mM 1a, 50 mM PHB buffer (respective pH value),

at 40 °C in a total volume of 200 µL (i.e. 150 µL enzyme mixture were added to 50 µL nucleoside mixture

with both mixtures containing the same PHB buffer concentration and a final total concentration of

1 mM 1a and 3 µg mL-1 TtPyNP). The assay was performed at 40 °C to ensure enzymatic activity during

the activity assay without denaturation during the reaction. From these reactions, samples of 50 µL

were withdrawn after 1, 2 and 4 min, quenched and analyzed as described above. All data regarding


7

residual activity were compared to the activity without any incubation. To obtain the half-life of

GtPyNP, activity data were fitted to the first-order exponential decay function

�� = ��,� �� (S1)

where �� is the rate constant [s-1] observed after an incubation time [min] (or [h]), ��,� is the

observed rate constant prior to incubation [s-1] and � is the mean catalyst lifetime [min] from which

the half-life �/� [min] can be obtained via

�/� = � ��2� (S2)

with definitions from above. Please see Table S2 for obtained half-lives and Figures S2 for the fits. For

pH 7−9, no significant loss in activity was observed within the 60 min incubation time. All raw and

transformed data for this experiment can be obtained free of charge from an external online

repository.[6]

Table S2. Half-lives of GtPyNP at 65 °C.

pH �/� [min-1] R2

7 >60 -

7.5 >60 -

8 >60 -

8.5 >60 -

9 >60 -

9.5 51.8 ± 9.2[a] 0.72

10 34.5 ± 6.7[b] 0.66

[a] fitted with ��,� = 4.2 s-1, [b] fitted with ��,� = 3.5 s-1

Figure S2. Half-life fits for GtPyNP at 65 °C and pH 9.5 and 10. Data were fitted to equation (S1).


8

The Michaelis-Menten kinetics of GtPyNP were analyzed using reaction mixtures with varying

substrate concentrations. Reactions were performed using 2.4−24 µg mL-1 GtPyNP (for 1a) or

0.6−1.4 µg mL-1 GtPyNP (for 1b) in 50 mM PHB buffer in a total volume of 200 or 500 µL with nucleoside

concentrations of 0.1, 0.25, 0.5, 1.0, 2.0 and 5.0 mM (for 1a) or 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM (for

1b), temperatures and pH values as indicated. The KM for phosphate was approximated by running

reactions of 2 mM 1a and 4.8 µg mL-1 GtPyNP in 3, 5, 10, 20 or 50 mM PHB buffer at 40 °C and pH 7.

All reactions were run in triplicate with samples drawn at variable time points as detailed in the

metadata files online. Samples were withdrawn, quenched and analyzed as described above. The

obtained data were fitted to the Michaelis-Menten equation according to

��,�� = �� [�]!" + [�] (S3)

where ��,�� is maximum observed rate constant (equal to �$��), �� is the observed rate constant,

[�] is the substrate concentration and KM is the Michaelis-Menten constant.

The KM for phosphate could not be determined with any degree of accuracy since the reaction system

is under thermodynamic control.[10] Therefore, decreases of activity at lower phosphate

concentrations than those tested would have been due to the lower equilibrium position of the

reaction and the corresponding greater contribution of the reverse reaction (glycosylation). At 3 mM

phosphate and 2 mM 1a (the lowest phosphate concentration tested), the equilibrium is only at 34%

conversion of 1a, rending experiments at even lower phosphate concentrations unfeasible. However,

the data in Figure S3E clearly show a KM of ca. 1 mM, easily letting us work at saturating conditions.

We only determined the KM for 1a at 40 °C (and not at 60 °C), since the KM for this substrate was

generally low and easily permitted us to work with substrate concentrations of >3 KM. Since the KM for

1a showed little pH-dependence and the KM for 1b was only minorly influenced by the reaction

temperature, we anticipated that 2 mM of 1a would always be in a saturating concentration regime.


9

Figure S3. Michaelis-Menten plots of GtPyNP with 1a (A and B), 1b (C and D) and phosphate (E). Data

were fitted to equation (S3).


10

The Eyring plots for GtPyNP-catalyzed phosphorolysis of 1a and 1b were obtained by running

reactions with 2 mM (for 1a) or 5 mM (for 1b) nucleoside and 0.5−25 µg mL-1 GtPyNP in 50 mM PHB

buffer at 30−65 °C and pH 7−10 in a total volume of 200 µL as indicated in the metadata files in the

externally hosted Supplementary Information. Reaction mixtures (120 µL) were preheated for 30 s at

the reaction temperature and the reaction was started by addition of analogously preheated enzyme

mix (80 µL). Samples were withdrawn after 20, 40 and 60 s and quenched in aqueous NaOH as

described above. The observed rate constant was calculated and the obtained data were fit to the

linearized Eyring and MMRT equations (S4) and (S5). For equation (S5), %� was set to 298 K. Please see

Figure S4 for the graphs and Table S3 for tabulated fit results. All raw data and transformations are

available from the externally hosted Supplementary Information.[2]

�� = �� %ℎ − Δ(‡)% + Δ�‡

) (S4)

�� = �� %ℎ − *Δ(+�‡ + Δ��‡% − %��)% , + *ΔS+�‡ + Δ��‡��% − ��%��) , (S5)

As Figure S3 details, the concentration of 1a selected for these experiments (2 mM) was in the

saturation regime across the pH range tested (KM = 0.62 mM at pH 7 and 0.89 mM at pH 9). Likewise,

phosphate was always present in saturating concentrations. However, 1b presented a more

challenging situation. While we could achieve near-saturating conditions at lower pH values (5 mM 1b

for KM = 3.1 mM), this was not possible anymore at higher pH values where the deprotonation of the

nucleobase increased the KM beyond the accessible concentrations (as 1b is poorly soluble). Thus, we

selected 5 mM for testing across the entire pH range. Since the KM for 1b only showed little

temperature dependence, we expect that our Eyring plots are only very minorly impacted by affinity

effects. Therefore, �� does not reflect �$�� for 1b at higher pH values, but still reliably provides its

temperature-dependence.


11


12

Figure S4. Eyring plots for GtPyNP-catalyzed phosphorolysis of 1a and 1b in PHB buffer. Data were fit

to equations (S4) and (S5), for the Eyring and MMRT graphs, respectively.

Table S3. Fit results of the kinetic data of GtPyNP-mediated phosphorolysis of 1a and 1b.

Substrate pH Model ./‡[a]

[kJ mol-1]

.0‡[a]

[J mol-1 K-1]

.12‡

[kJ mol-1 K-1] R2 AIC

1a

7 MMRT 92.2 ± 15.2 66.3 ± 49.3 -1.9 ± 0.7 0.86 -111.4

Eyring 49.9 ± 3.5 -71.8 ± 10.8 - 0.84 -105.8

7.5 MMRT 89.9 ± 14.6 59.4 ± 47.7 -1.6 ± 0.6 0.89 -114.6

Eyring 54.8 ± 3.3 -55.0 ± 10.2 - 0.88 -111.0

8 MMRT 101.6 ± 14.8 97.3 ± 48.3 -1.9 ± 0.7 0.90 -113.4

Eyring 59.0 ± 3.4 -41.9 ± 10.6 - 0.88 -107.4

8.5 MMRT 96.9 ± 13.3 82.8 ± 43.4 -1.7 ± 0.6 0.92 -124.5

Eyring 58.6 ± 3.0 -42.2 ± 9.3 - 0.91 -118.6

9 MMRT 96.9 ± 12.6 83.1 ± 41.1 -1.7 ± 0.6 0.93 -127.8

Eyring 60.0 ± 2.9 -37.2 ± 9.1 - 0.91 -121.5

9.5 MMRT 99.6 ± 12.5 92.1 ± 40.1 -1.9 ± 0.5 0.92 -128.8

Eyring 56.5 ± 3.0 -48.6 ± - 0.90 -119.5

10 MMRT 101.4 ± 12.6 97.9 ± 41.1 -2.4 ± 0.6 0.90 -125.1

Eyring 50.3 ± 3.2 -68.7 ± 10.0 - 0.87 -112.2

1b

7 MMRT 62.5 ± 14.8 -3.3 ± 48.2 -1.2 ± 0.6 0.79 -106.7

Eyring 36.1 ± 3.3 -89.4 ± 10.4 - 0.77 -105.7

7.5 MMRT 73.9 ± 13.4 35.3 ± 43.6 -1.8 ± 0.6 0.80 -112.8

Eyring 33.0 ± 3.3 -97.9 ± 10.2 - 0.75 -105.9

8 MMRT 54.0 ± 17.1 -29.0 ± 55.7 -1.0 ± 0.7 0.66 -96.3

Eyring 30.5 ± 3.8 -105.6 ± 11.7 - 0.66 -96.7

8.5 MMRT 68.1 ± 12.0 14.5 ± 39.2 -1.4 ± 0.5 0.85 -126.4

Eyring 36.7 ± 2.8 -87.7 ± 8.7 - 0.83 -121.9

9 MMRT 68.4 ± 13.4 13.8 ± 43.5 -1.3 ± 0.6 0.84 -117.8

Eyring 39.5 ± 3.0 -80.4 ± 9.4 - 0.83 -115.3

9.5 MMRT 67.0 ± 10.1 5.8 ± 32.7 -1.6 ± 0.4 0.85 -117.8

Eyring 30.1 ± 2.6 -114.2 ± 8.2 - 0.80 -115.3

10 MMRT 64.1 ± 11.8 -6.9 ± 38.6 -1.4 ± 0.5 0.84 -117.9

Eyring 33.7 ± 2.9 -105.9 ± 9.1 - 0.81 -113.6

[a] The Δ(‡ and Δ�‡ for the MMRT fits represent the corresponding value at %� = 298 K, i.e. Δ(�345‡

and Δ��345‡.


13

Figure S5. Charges of the buffer components employed in this study over the explored pH range. The

individual charges were calculated with the Henderson-Hasselbach equation for pKa values of 7.2, 8.0

and 9.2.

The theoretical optimum temperatures of the GtPyNP-catalyzed phosphorolysis of 1a and 1b was

obtained via equation (S6).[17]

%�� = Δ(+�‡ − Δ��‡%�−Δ��‡ − ) (S6)

with definitions from above. The optimal temperature for the phosphorolysis of 1a at pH 7, %��,67 =

75 °C, was calculated with %� = 298 K and Δ��‡ = -1.9 [kJ mol-1 K-1] and the corresponding value for 1b,

%��,68 = 79 °C, with %� = 298 K and Δ��‡ = -1.2 [kJ mol-1 K-1].

Figure S6. Theoretical optimum temperatures of the GtPyNP-catalyzed phosphorolysis of 1a and 1b at

pH 7.


14

Scheme S1. Mechanism of pyrimidine nucleoside phosphorylase-catalyzed phosphorolysis as indicated

from published crystal structures (e.g. 1uou or 2wk6).


15

Scheme S2. Energy profile for GtPyNP-catalyzed nucleoside phosphorolysis at 298 K and pH 7. The free

enthalpies of the products were calculated from known equilibrium constants (0.132 for 1a and 0.109

for 1b at 298 K)[10] and the transition state free enthalpies were obtained via the Gibbs-Helmholtz

equation and the values listed in Table S3 for pH 7.

Supplementary references

[1] A. Brand, L. Allen, M. Altman, M. Hlava, J. Scott, Learn. Publ. 2015, 28, 151–155.


[3] K. Szeker, X. Zhou, T. Schwab, A. Casanueva, D. Cowan, I. A. Mikhailopulo, P. Neubauer, J. Mol.

Catal. B Enzym. 2012, 84, 27–34.

[4] F. Kaspar, R. T. Giessmann, N. Krausch, P. Neubauer, A. Wagner, M. Gimpel, Methods Protoc.

2019, 2, 60.

[5] E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M. R. Wilkons, R. D. Appel, A. Bairoch, in

Proteomics Protoc. Handb. (Ed.: J. Walker), Humana Press, 2005, pp. 571–607.


[7] F. Kaspar, R. T. Giessmann, S. Westarp, K. F. Hellendahl, N. Krausch, I. Thiele, M. C. Walczak, P.

Neubauer, A. Wagner, ChemBioChem 2020, 21, 2604.


[9] A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, 1979.

[10] F. Kaspar, R. T. Giessmann, P. Neubauer, A. Wagner, M. Gimpel, Adv. Synth. Catal. 2020, 362,

867–876.

[11] D. H. Kim, K. F. Faull, A. J. Norris, C. D. Eckhert, J. Mass Spectrom. 2004, 39, 743–751.

[12] J. K. Hobbs, W. Jiao, A. D. Easter, E. J. Parker, L. A. Schipper, V. L. Arcus, ACS Chem. Biol. 2013,

8, 2388–2393.

[13] J. K. Hobbs, C. Shepherd, D. J. Saul, N. J. Demetras, S. Haaning, C. R. Monk, R. M. Daniel, V. L.


16

Arcus, Mol. Biol. Evol. 2012, 29, 825–835.

[14] M. W. van der Kamp, E. J. Prentice, K. L. Kraakman, M. Connolly, A. J. Mulholland, V. L. Arcus,

Nat. Commun. 2018, 9, 1177.

[15] R. S. Firestone, S. A. Cameron, J. M. Karp, V. L. Arcus, V. L. Schramm, ACS Chem. Biol. 2017, 12,

464–473.

[16] M. E. Peterson, R. M. Daniel, M. J. Danson, R. Eisenthal, Biochem. J. 2007, 402, 331–337.

[17] V. L. Arcus, E. J. Prentice, J. K. Hobbs, A. J. Mulholland, M. W. Van der Kamp, C. R. Pudney, E. J.

Parker, L. A. Schipper, Biochemistry 2016, 55, 1681–1688.

[18] M. E. Peterson, R. Eisenthal, M. J. Danson, A. Spence, R. M. Daniel, J. Biol. Chem. 2004, 279,

20717–20722.

[19] R. S. Phillips, S. Craig, A. Kovalevsky, O. Gerlits, K. Weiss, A. I. Iorgu, D. J. Heyes, S. Hay, ACS

Catal. 2020, 10, 1692–1703.

[20] H. B. L. Jones, R. M. Crean, A. Mullen, E. G. Kendrick, S. D. Bull, S. A. Wells, D. R. Carbery, F.

MacMillan, M. W. van der Kamp, C. R. Pudney, Biochemistry 2019, 58, 2362–2372.

[21] H. B. L. Jones, R. M. Crean, C. Matthews, A. B. Troya, M. J. Danson, S. D. Bull, V. L. Arcus, M. W.

van der Kamp, C. R. Pudney, ACS Catal. 2018, 8, 5340–5349.

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Chem. Soc. 2019, 141, 11745–11748.

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pH-Independent Heat Capacity Changes during Phosphorolysis