Table 1.

Direct evidence that the enolization equilibrium differs between Rubisco forms, and that decarboxylation and deoxygenation are negligible.

Questions raised by Cummins et al. (2018)	Answer (yes/no)	Experimental evidence	References
Is the decarboxylation rate of importance?	No	1. The partitioning (catalysis:decarboxylation) of the 6-carbon intermediate when it is fed to the enzyme has been shown to be at least 95:5.	Lorimer et al., 1986
		2. Should decarboxylation be substantial, we should observe a small 12C/13C kinetic isotope effect (13V/K) during carboxylation. In fact, 13V/K is given by αeq·(1 + cα7)/(1 + c) where αeq is the equilibrium isotope effect of carboxylation, α7 is the kinetic isotope effect of decarboxylation and c the commitment to catalysis (c = k8b/k7). CO2 addition on sugars forming a ternary C atom favors 13C by about 3%0 so that αeq is about 0.997. A value of c = 1 gives a fractionation within 0.997-1.011 for possible values of α7 between 1 and 1.030 (feasible range for a 12C/13C kinetic isotope effect). It is therefore impossible to match the observed isotope effect in most organisms (13V/K ≈ 1.030 in higher plants) unless assuming extremely high values of the isotope effect for decarboxylation (about 1.070).	O'Leary and Yapp, 1978; Roeske and O'Leary, 1984, 1985; Rishavy and Cleland, 1999
Is enolization variable and thus can KR (and γc) change a lot between Rubiscos?	Yes	1. A typical example is Rhodospirillum rubrum, which does not fit the empirical linearization used by Cummins et al. (2018). In fact, the intrinsic 1H/2H isotope effect (RuBP deuterated in H3) on maximal velocity (DV) when enolization becomes rate-limiting (at low pH) is clearly lower in R. rubrum than in spinach; in addition, the isotope effect at limiting RuBP (DV/K) is unity in R. rubrum but increases at low pH, contrary to what is observed in spinach. The enzyme of R. rubrum can also easily exchange the H3 proton with the solvent.	Saver and Knowles, 1982; Van Dyk and Schloss, 1986
		2. There are considerable differences in the ability to carboxylate xylulose-1,5-bisphosphate (C3 stereoisomer of RuBP) between higher plants, prokaryotes and red algae, showing that the mechanistic constraints on H3 abstraction and thus stereochemistry of enolization differ between Rubisco forms.	Pearce, 2006
Is the deoxygenation rate of importance?	No	1. O2 addition forms a peroxide. In general, oxygenation to a peroxide is irreversible and thus deoxygenation of a peroxide is extremely unlikely.	Frankvoort, 1978; Lorimer, 1981
		2. Should the peroxide be deoxygenated, deoxygenation would not be the reverse of oxygenation because the spin-forbidden character of oxygenation requires excited chemical forms that are unlikely to be reformed. In practice, going backwards from the peroxide to the enediolate is strongly thermodynamically disfavored.	Jonsson, 1996; Bathellier et al., 2018
		3. As with 13C (above), the 16O/18O isotope effect during oxygenation (18V/K ≈ 1.021) indicates that an important commitment to deoxygenation is not credible.	Guy et al., 1993