only a few enzymes have been characterized. 



Variations in subunit sequence and structure are only one 

 parameter that might be used for diagnosis. A reflection of 

 structure is the strength of L/S binding. Microcolorimetry, using 

 purified enzymes, could be used to measure this parameter. Along 

 with tight-binding of subunits, inhibitor binding and "misfiring" 

 of the enzyme should be of value. Bound inhibitors (e.g., CABP and 

 CA1P) and misfired products (e.g., xylulose bisphosphate) could be 

 measured by HPLC after destroying the enzyme. Although much is 

 known about the enzyme, little is known about its structural and 

 biochemical features in most organisms; apart from higher plants 

 (where probably a couple of hundred Rubisco enzymes are 

 characterized) , only one or two model species are studied in other 

 groups. Yet another aspect of the problem is the activation of 

 inactive enzymes by Rubisco activase. We need to know more about 

 the biochemistry of activation, the activase isogenes, and isoforms 

 of this enzyme. 



One of the most relevant parameters in understanding the 

 regulation of Rubisco activity is the so-called specificity factor; 

 namely, the preference of the enzyme to accept COp over O2 as the 

 substrate. The ratio of specificity of oxygenase and carboxylase 

 activities for Rubisco has been evaluated in only a narrow range of 

 organisms, few of them relevant to the bulk of C0« flux through the 

 biosphere. This flux will be sensitive to the specificity factor. 

 The specificity factor, x , has changed during evolution from 10 

 (photosynthetic bacteria) , to 50 (cyanobacteria) , to 70-100 (higher 

 plants) . Without a suitable site-directed mutagenesis system in 

 which to study how the primary structure of the enzyme is related 

 to the specificity factor, comparative molecular biological 

 approaches are needed. Adaptations of Rubisco to ecological niches 

 appear likely, and the study of unique eukaryotic systems, which 

 have different catalytic subunit sequences, provides a basis for 

 such an approach. 



Surveys of sequence variation are needed, coupled with 

 measurements using mass spectrometry (simultaneous O2 uptake and 

 CO- evolution) , in a wide range of relevant terrestrial and marine 

 organisms. 



(2) Participation of carbonic anhydrase in C0 2 < > HgO 



II-4 



