# A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart
## 4 Discussion  
### Presence or Absence of RbcS External to Active Site May Influence KIE  
Rubisco KIES have also been used to support conclusions gleaned from non-isotopic kinetic parameters, both to better understand the reaction mechanism and to offer complementary data to traditional measurements, but our results belie an easy interpretation within that existing framework. The dominant theory in this field posits that rubisco specificity is positively correlated with the CO\({}_{2}\) KIE because of an observed increase in carbon isotope fractionation, but not oxygen isotope fractionation, with specificity [15; 25]. This argument originates from studies of deuterium (D) isotope effects on enzymatic reaction rates, which have been traditionally performed because deuterium displays a much larger (and easier to measure) KIE due to the large relative mass difference between D and its major isotope, H, in comparison to other rare isotopes such as \({}^{13}\)C vs. \({}^{12}\)C or \({}^{15}\)N vs. \({}^{14}\)N [65]. These foundational experiments have led to the conclusion that the isotope effect is determined at the rate-limiting step at the transition state, and small asymmetries in the transition state caused by transition state structure will cause small variations in the isotope effect [65; 66]. Applied to rubisco, [15] proposed that the inherent difficulty in binding a 'featureless' CO\({}_{2}\) vs. O\({}_{2}\) molecule has caused natural selection in the transition state, where rubiscos that maximize the structural difference in transition states for carboxylation vs. oxygenation are able to be more specific. That then causes a trade-off where greater resemblance to the final carboxy ketone product causes the product to also be tightly bound, leading to a higher S\({}_{\text{C/O}}\) correlating with a lower V\({}_{\text{C}}\), but also a prediction that the intrinsic KIE for CO\({}_{2}\) addition (but not O\({}_{2}\) addition) should increase as the transition state becomes more product like, i.e., higher-specificity rubiscos should have higher KIES, which is indeed what the data at the time supported [15]. This has also led to the conclusion that rubisco is actually perfectly optimized for the time and places where it is found today, precluding any opportunity to use rubisco engineering to achieve increased biomass yields [15]. However, new CO\({}_{2}\) KIE measurements that do not show a correlation with specificity are empirically questioning this conclusion (Figure 1B). Prior studies [37] have pointed out that the spread in KIE data, particularly at high specificity, cannot easily be described by a simple inverse relationship or linear regression. Indeed, our Form I\({}^{\prime}\) measurement lies below the original regression line (dashed line in Figure 1B) proposed in [15]; its KIE is effectively too low given what one would predict via its specificity. However, although an increasing spread in CO\({}_{2}\) KIE becomes apparent as more rubiscos are measured, they cannot directly address the dominant theory because of the general dearth of O2 KIE measurements. In addition, specificity is typically not reported in the same study with KIE (see notes in Tables S2 and S3), so some of the spread in Figure 1B may be due to uncertainties in the true specificity for the given rubisco measured. Therefore, additional paired measurements of CO2 and O2 KIEs with specificity are necessary before a new theory relating isotopic and non-isotopic kinetics can be proposed; more data are needed to decide between potential theories. In addition, this transition state optimization theory is based on the assumption that it is the active site (which binds the intermediary carboxylation or oxygenation product) that concurrently affects both specificity and KIE, so the naive assumption is that the absence or presence of the small subunit, which does _not_ contain the active site, should not affect KIE.