Hello! I'm currently using the IntEnzyDB (https://intenzydb.accre.vanderbilt.edu/kinetics/list) for a machine learning project. During this process, I noticed that for some observations in the Kinetics Data table, the substrate_kinetics column contains multiple substrates in the form of a merged string separated by ";", which is presumably associated with multi-substrate enzymatic reactions. However, the Km entry of the wildtype enzyme sequence (column Km Wildtype) only shows one value, and the same goes for kcat. I've checked some of these reactions in UniprotKB, and there are separate Km values for different substrates. I've looked into the published paper, but I can't seem to find any relevant information. As I understand that the kinetics of multi-substrate systems could be complicated, I'd like to ask whether someone could kindly provide me with some guidance on how to interpret those entries in the database. I really appreciate your time and support!
In my web browser, there is a separate line for each substrate of a given enzyme. For example, for P39462 (NAD-depenedent alcohol dehydrogenase) of Sulfolobus sulfataricus, there is a line for NAD+ (the common substrate for all the reactions) and separate lines for some second substrates, such as 1-heptanol and isobutyraldehyde. Thus, the Km for each substrate is listed on a separate line.
As for the kcat, there should be only one kcat for the reaction between any pair of substrates, in principle, for a 2-substrate enzyme, rather than separate kcats for each of the two substrates. However, people often measure the kcat for one substrate at a fixed concentration of the other (not best-practice), which may result in different kcats for the 2 substrates if the fixed substrate concentration is not fully saturating or there is substrate inhibition by it.
Adam B Shapiro, thank you for your clarification on kcat!
As for Km, substrates are indeed listed on separate lines for the enzyme that you mentioned, but for some others, substrates are clumped together and separated by semicolon. Take P12268 for example, its 2 substrates are listed as "NAD+;IMP" in the substrate_kinetics column, and the Km Wildtype column only has 1.7e-05 as its only value (please see attached image). Checking in the UniprotKB, the Km of P12268 for NAD+ is 9.3e-06 and for Inosine 5'-phosphate is 3.2e-05 (https://www.uniprot.org/uniprotkb/P12268/entry).
I'm a bit perplexed by this behavior, so I'd really appreciate it if you could offer your perspective on this. Thank you!
The database was probably created by automated text mining of the literature, with imperfect results. I've no idea why the two substrates are not on separate lines.
Below are the references at the UniProt site for P12268 (IMPDH2) associated with the kinetics data.
From the abstract of Hager et al (1995) comes the following:
"Type I and II IMPDH had kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM."
From the abstract of Carr et al (1993) comes the following:
"Substrate affinities were similar for types I and II with Km values of 18 and 9.3 microM, respectively, for IMP, and 46 and 32 microM, respectively, for NAD.kcat values were 1.5 and 1.3 s-1 at 37 degrees C for types I and II, respectively."
Source NAD Km IMP Km kcat
Hager 32 µM 9 µM 1.4 s-1
Carr 32 µM 9.3 µM 1.3 s-1
More references with kinetic data for this enzyme can be found in the BRENDA database here:
https://brenda-enzymes.org/enzyme.php?ecno=1.1.1.205&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Homo+sapiens&show_tm=0#KM%20VALUE%20[mM]
The values of kinetic measurements depend on the conditions of the measurement, among other things, so expect considerable variation between reports.
- Badges
- Science topic
- Similar topics
- Philosophy
- Esthetics
- Interpretation