Experimental data file PPK kinetics

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Kinetic measurements 31 P NMR measurements

Figure 5 D

polyP synthesis
ADP Concentration was determined discontinously with PK-LDH Assay at 37°C
Assay at 70°C	Note	PPK [µg]	ATP [mM]	Mg[mM]	TRIS Buffer [mM]	Mean specific Activity [mU/mg]	SD [mU/mg]
polyP synthesis Fig. 4D	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	10	4	4	50	186.85847506068504	33.54056004743149
	PPK: heteromeric PPK	10	3	4	50	222.9983136214427	42.2830462076463
		10	2	4	50	214.4096036251298	8.691481264394412
		10	1.5	4	50	147.54072262165607	16.21818411516726
		10	1	4	50	94.36872768556537	4.2920201614784155
		10	0.75	4	50	48.33800832258233	12.79514768572167
		10	0.5	4	50	25.964760925092072	0
		10	0.25	4	50	11.212070065444362	9.272781989937625
		10	0.1	4	50	5.815863204430399	0.13905801546534644
		10	1.0E-6	4	50	0	0


ATP synthesis
ATP Concentration was determined discontinously with HK-G6PDH Assay at 37°C
Assay at 70°C
ADP in saturation	Note	PPK [µg]	polyP45 [µM]	ADP [mM]	Mg[mM]	TRIS Buffer [mM]	Mean specific Activity [U/mg]	SD [U/mg]
ATP synthesis Fig. 4E	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	5	100	20	10	50	12.357054164123934	0.16987130414753718
	PPK: heteromeric PPK	5	75	20	10	50	11.912024954492345	0.2303815306587484
		5	50	20	10	50	9.137380918996133	0.13969432049383024
		5	25	20	10	50	2.825754381091484	0.08774178608431836
		5	15	20	10	50	1.2856344116727096	0.2988961645549357
		5	10	20	10	50	0.5291337894640746	0.1040952094210284
		5	7.5	20	10	50	0.2039563948923764	0.18149234330780456
		5	2.5	20	10	50	0.03708232040179955	0.16243929902202603
		5	1.0E-6	20	10	50	0	0

Assay at 70°C
polyP in saturation	Note	PPK [µg]	polyP45 [µM]	ADP [mM]	Mg[mM]	TRIS Buffer [mM]	Mean specific Activity [U/mg]	SD [U/mg]
ATP synthesis Fig. 4F	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	5	100	30	10	50	2.145481576556152	0.03078524160675172
	PPK: heteromeric PPK	5	100	20	10	50	2.7641831567893242	0.019926651161207454
		5	100	10	10	50	2.7260833985444215	0.08702136113778439
		5	100	8	10	50	2.5875283391638906	0.11740873097010723
		5	100	6	10	50	2.339370500532498	0.048739013645021936
		5	100	4	10	50	2.0576400268141253	0.09302101674322075
		5	100	2	10	50	1.2699442338511673	0.10161127423823689
		5	100	1.0E-6	10	50	0	0


polyP synthesis
ADP Concentration was determined discontinously with PK-LDH Assay at 37°C
	Note	PPK [µg]	ATP [mM]	Mg[mM]	TRIS Buffer [mM]	Mean specific Activity [mU/mg]	SD [mU/mg]
ATP degradation Fig. 4F	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	10	8	4	50	31.916275742245677	9.672528614336864
	PPK: homomeric PPK	10	6	4	50	35.770179787637396	10.431488660680795
		10	4	4	50	35.302105329227054	5.717076422687372
		10	3	4	50	32.59323828266209	6.134021034609912
		10	2	4	50	20.639495017595344	5.306827020552118
		10	1.5	4	50	14.123709232411553	5.155945965449727
		10	1	4	50	8.75407890829948	6.212583182219284
		10	1.0E-7	4	50	1.0E-7	0

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PPK
polyP synthesis
Figure 5B
70°C	Note	PPK [µg]	ATP [mM]	Mg[mM]	TRIS Buffer [mM]	D2O [%]
polyP synthesis Fig. 5B	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	20	2	4	50	10
	PPK: heteromeric PPK
	Start by adding the enzyme

Time	ADP	ATP
min	Normalized sum of integrals	Normalized sum of integrals
0	0.01892	0.98108
30	0.0527	0.9473
60	0.06735	0.93265
90	0.08977	0.91023
120	0.10178	0.89822
150	0.17087	0.82913
180	0.16846	0.83154
210	0.20237	0.79763
240	0.20301	0.79699
270	0.20774	0.79226
300	0.20323	0.79677
330	0.23312	0.76688
360	0.2079	0.7921

Figure 5C
70°C	Note	PPK [µg]	ATP [mM]	Mg[mM]	PEP [mM]	Sso_PK [µg]	TRIS Buffer [mM]	D2O [%]
polyP synthesis with ATP Recycling Fig. 5C	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	20	2	20	10	8	50	10
	PPK: heteromeric PPK
	Start by adding the enzyme
	PK: pyruvate kinase (S. solfataricus)
	PEP_phosphoenolpyruvate

Time	Pi	PEP	ADP	ATP	CP3	PolyP
min	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals
0	-0.00147	0.81665	0.0049	0.17691	0.00465	0.00237
30	0.03028	0.81052	0.00337	0.16816	-0.01256	-0.00521
45	0.04011	0.78517	0.0029	0.1728	-0.00981	0.00697
60	0.034	0.77035	0.00426	0.1692	0.00709	0.01407
75	0.05287	0.7661	0.01387	0.17401	-0.00704	-0.00175
90	0.04819	0.74317	-0.00432	0.19362	0.00806	0.00916
105	0.06545	0.73028	-3.74834E-4	0.15717	-0.00904	0.02338
120	0.08444	0.69451	0.00119	0.18916	-0.00476	0.03278
135	0.0873	0.66241	0.01363	0.17441	0.00267	0.05668
150	0.09386	0.65539	0.00842	0.18967	3.2796E-4	0.05323
165	0.1007	0.61547	0.01345	0.18257	0.00862	0.0853
180	0.14588	0.59842	-0.01519	0.19394	-0.01244	0.0826
195	0.13786	0.54622	7.86316E-4	0.18825	0.02867	0.10446
210	0.14445	0.52523	-0.00669	0.18898	0.02091	0.13684
225	0.1651	0.49868	-0.00171	0.17346	0.0232	0.15662
240	0.16943	0.45959	-0.00531	0.19545	0.00196	0.18799
255	0.16837	0.44191	-0.00669	0.19489	0.0211	0.18235
270	0.16718	0.37605	0.01706	0.21364	0.02893	0.2026
285	0.19536	0.34596	0.00989	0.18986	0.03421	0.23048
300	0.18124	0.32773	0.01016	0.1887	0.0559	0.23889
315	0.19123	0.26902	0.0205	0.19685	0.05821	0.26972
330	0.20096	0.25649	0.00372	0.21571	0.06021	0.28146
345	0.22883	0.18181	0.02851	0.20659	0.06548	0.2991
360	0.22958	0.18506	0.01006	0.19344	0.07367	0.31573
375	0.26695	0.13257	0.00797	0.1952	0.08619	0.32817
390	0.24147	0.14044	0.02335	0.20064	0.08634	0.31609
405	0.28148	0.09553	0.00557	0.18483	0.08412	0.3389
420	0.26903	0.0665	0.00786	0.21212	0.10937	0.33968
435	0.26563	0.04301	0.03364	0.20263	0.11567	0.35688
450	0.27775	0.00654	0.02375	0.23748	0.12146	0.34947
465	0.29781	0.01392	0.01676	0.19547	0.14938	0.33498
480	0.30106	0.00766	0.02006	0.19771	0.15528	0.31916
495	0.31118	-0.00676	0.02239	0.18429	0.15721	0.3425
510	0.31985	0.01556	0.02374	0.15118	0.16048	0.32857
525	0.34179	-0.01028	0.04048	0.15003	0.18356	0.30287
540	0.34511	-0.00502	0.0293	0.1674	0.20692	0.26138
555	0.34967	0.00948	0.0266	0.14213	0.19591	0.2774
570	0.37537	0.01222	0.01713	0.16086	0.20269	0.22816
585	0.36759	0.01097	0.03877	0.15069	0.20852	0.22377
600	0.37538	0.01922	0.02784	0.16361	0.21159	0.19761
615	0.38543	-0.00299	0.04506	0.14579	0.21499	0.20065
630	0.39201	0.01386	0.04492	0.15281	0.22809	0.17283
645	0.39913	0.00875	0.03468	0.12933	0.2429	0.17006
660	0.42847	0.00818	0.03455	0.14615	0.24613	0.13184
675	0.44102	-0.02073	0.03358	0.1592	0.2548	0.12463
690	0.44114	0.0093	0.04602	0.13625	0.2435	0.11315
705	0.46733	-0.00378	0.04119	0.1444	0.2487	0.09398
720	0.44433	0.03569	0.03917	0.14117	0.23986	0.07928
735	0.47919	-0.00492	0.04004	0.13271	0.2558	0.07949

Figure 5A
70°C	Note	PPK [µg]	ADP [mM]	Mg[mM]	polyP45 [µM]	TRIS Buffer [mM]	D2O [%]
ATP synthesis Fig. 5A	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	5	10	10	500	50	10
	PPK: heteromeric PPK				Are responsible to get a good signal to noise ratio for polyP!
	Start by adding the enzyme

Time	ADP	ATP	PolyP	CycloTriP	Pi
min	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals
0	0.30865	0.01406	0.61802	0.03333	0.02595
15	0.28627	0.02256	0.62482	0.04238	0.02397
30	0.27917	0.03868	0.6039	0.05452	0.02373
45	0.26313	0.0604	0.58732	0.06414	0.025
60	0.25136	0.08221	0.55935	0.07407	0.03301
75	0.23732	0.10789	0.53454	0.08773	0.03253
90	0.2127	0.13972	0.5123	0.09741	0.03787
105	0.20422	0.16522	0.47875	0.11064	0.04117
120	0.18425	0.18749	0.46015	0.1221	0.04601
135	0.17489	0.2048	0.43529	0.13451	0.05051
150	0.17137	0.20089	0.42614	0.14418	0.05741
165	0.17642	0.1993	0.40855	0.14962	0.06611
180	0.16701	0.20995	0.39321	0.15699	0.07285
195	0.16894	0.20907	0.37817	0.1665	0.07732
210	0.16387	0.20591	0.37887	0.17538	0.07596
225	0.16217	0.21862	0.35201	0.18434	0.08286
240	0.15964	0.22061	0.34668	0.19462	0.07845
255	0.15439	0.23156	0.32197	0.20493	0.08714
270	0.14371	0.24297	0.31015	0.21387	0.0893
285	0.14146	0.25179	0.29258	0.2222	0.09196
300	0.13375	0.26047	0.27469	0.23056	0.10054
SI Fig. 12 B
PEP Hydrolysis at 70°C
	Note	PPK [µg]	Mg[mM]	PEP [mM]	TRIS Buffer [mM]	D2O [%]
PEP hydrolysis (SI part)	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	20	20	10	50	10
	PPK: heteromeric PPK


Time	Pi	PEP
min	Normalized sum of integrals	Normalized sum of integrals
0	0	1
15	0	1
30	0.02586	0.97414
45	0.01195	0.98805
60	0.03566	0.96434
75	0.03547	0.96453
90	0.04006	0.95994
105	0.05494	0.94506
120	0.07174	0.92826
135	0.06377	0.93623
150	0.0778	0.9222
165	0.05924	0.94076
180	0.08255	0.91745
195	0.08968	0.91032
210	0.08617	0.91383
225	0.1036	0.8964
240	0.09984	0.90016
255	0.11204	0.88796
270	0.12069	0.87931
285	0.13198	0.86802
300	0.12182	0.87818
315	0.14156	0.85844
330	0.12099	0.87901
345	0.12352	0.87648
360	0.15004	0.84996
375	0.15912	0.84088
390	0.14704	0.85296
405	0.15243	0.84757
420	0.16903	0.83097
435	0.17021	0.82979
450	0.17546	0.82454
465	0.16391	0.83609
480	0.18666	0.81334
495	0.18035	0.81965
510	0.1936	0.8064
525	0.19977	0.80023
540	0.20492	0.79508
555	0.19	0.81
570	0.21225	0.78775
585	0.21023	0.78977
600	0.22254	0.77746
615	0.21478	0.78522
630	0.23527	0.76473
645	0.23662	0.76338
660	0.25262	0.74738
675	0.23702	0.76298
690	0.2554	0.7446
705	0.26061	0.73939
720	0.25646	0.74354
735	0.26042	0.73958
750	0.27352	0.72648
765	0.28802	0.71198
780	0.27373	0.72627
795	0.28274	0.71726
810	0.28819	0.71181


Figure 5A
70°C	Note	PPK [µg]	ADP [mM]	Mg[mM]	polyP45 [µM]	TRIS Buffer [mM]	D2O [%]
ATP synthesis Fig. 5A	50 mM TRIS/HCl pH 7.4 with 50 mM KCl	20	10	10	500	50	10
	PPK: heteromeric PPK				Are responsible to get a good signal to noise ratio for polyP!
	Start by adding the enzyme

Time	Pi	ADP	ATP	cP3	polyP
min	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals	Normalized sum of integrals
0	0.034815681685196026	0.3227033352837917	0	0.03613224107665301	0.5836746635459333
15	0.04836174085717587	0.26839550138488116	0.0538145312424054	0.0526977241855378	0.5497503278832155
30	0.05710512853421035	0.1798585236414097	0.1929181145576874	0.07333033817722899	0.47352793681350513
45	0.060613082714388374	0.12116180362096	0.27054997018018606	0.08498080261360154	0.42528818287991954
60	0.0661621892056738	0.10987059929580306	0.2931625881167062	0.09162285051288233	0.40247112991778033
75	0.06985257954097543	0.09999540734594138	0.30534574063918857	0.1007861488557031	0.38933366378279854
90	0.07489436866683041	0.10397893629224286	0.2983778616552453	0.11848584440846105	0.37061105846782116
105	0.07410687592711532	0.10841940357292155	0.3077108024174846	0.1266524769592133	0.3466918156066605
120	0.0875828642788945	0.10893855824702871	0.30115952673789087	0.13342471740044493	0.33161502535490345
135	0.08884252669899291	0.09766947737393289	0.3029630971694309	0.14235471864984528	0.32317710647277825
150	0.09188429534334185	0.10425575547964328	0.3027000442177048	0.14873446617889244	0.31234006669156333
165	0.09576060222665675	0.09292055530979929	0.31297254564211113	0.16327860782149722	0.29498729341397883
180	0.10367592188368228	0.10493194752946158	0.30364704444153773	0.16383675552006402	0.2782195776642041