Lactate dehydrogenase: Structure and Function

Aims

In this exercise you are going to study the tertiary structure of an interesting enzyme, L-lactate dehydrogenase (LDH, E.C.1.1.1.27). LDH is the enzyme that you will purify from pig heart tissue and then characterize during the lab exercise. This tutorial also trains you in finding relevant information stored in databases on the web, which is an important part of practical biochemical work. Gathering as much background information as possible can help you in experiment planning and save time and effort, as well as guide you to perform better experiments.

You shall answer the following questions:

  • Which reaction is catalyzed by LDH in vivo?

LDH exists in different isoenzyme forms. To analyze if a patient has suffered from a heart attack you can look for a specific LDH isoenzyme present mainly in heart tissue and is leaked into the blood during an infarct.

  • Which is the LDH isoenzyme found in heart tissue?

You will study the pig heart LDH since it is (naturally) difficult to get hold of human hearts and the pig enzyme is a relatively good model.

  • How similar are the human and the pig heart LDH enzymes?
  • What is the (calculated) isoelectric point of the pig LDH?
    • How can this information aid you in planning for the purification?
  • What is the molecular weight?
  • How does the published value correlate with your own experimental results? Discuss possible differences in the LDH lab report.
  • What has been reported earlier on kinetic properties as described by Km and kcat? What about specific activity? Is data for the pig enzyme available (pig = "Sus scrofa" in Latin)?
  • Are previously reported values consistent? If not, comment. How do these values compare to your own results? Discuss in the LDH lab report.

Look at the tertiary structure of LDH

Use a structure file that also contains a bound cofactor (if available in the PDB). Analyze how the cofactor is bound at the active site and which residues in the protein that interacts with it.

  • Is it possible to deduce the binding site for the second substrate? Draw a sketch of the interactions in the active site. Append with the LDH lab report.

Arg170 and His194 are important for its catalytic function.

  • Speculate on possible reasons for this.

Arg107, Thr249, Val32 and Arg100 appear to be important for substrate binding (pig LDH numbering).

  • Can this be explained from the interactions these residues make in the enzyme? Comment in the LDH lab report.
  • Compare the structures of the human and the pig enzymes. Do they differ in any significant way?
  • Finally, is the pig enzyme a good model for the human counterpart? Motivate your answer. 

Work flow

Check in the text book what is said about LDH. After this, go to the database ENZYME that can provide general information about enzymes. Use the EC number as search query. ENZYME also provide links to related databses which may be useful for your work.

Report

  • Include all relevant information in the LDH lab report. Some may fit in the introduction and other in the discussion part. Use your own judgement.
  • Append relevant figures.