New enzymes

1

• New enzymes
• Adenosylhomocysteinase
• Thermolysin

2

• New enzymes
• Lachrymatory factor synthase

3

• New enzymes RNase Z

4

• New enzymes RNase Z
• Other ribonucleases
• RNase A (3.1.27.5) 134 aa in human. Pancreatic
  enzyme. His12 and His119 are involved as acid and
  base. A cyclic phosphodiester is formed.
• RNase D (3.1.26.3) 123 aa catalytic domain in
  E.coli. Exonuclease activity trimming RNase P
  products down to the 3 CCA end.
• RNase P 377 bp mRNA 20 kDa protein. Ribozyme.
  Cleavage of the primary RNA transcript at the 5
  side of the tRNAs (prokaryotes). Active at 60 mM
  Mg2.
• RNase III 118 aa catalytic domain (A.aeolicus).
  Mg2 dependent. Creates dimer and cleaves dsRNA.

5

• New enzymes
• RNase Z Cleavage of the primary RNA transcript
  (ssRNA, but folded) at the 5 side of the tRNAs
  (eukaryotes).

6

• New enzymes RNase Z
• Test quaternary structure (monomer / dimer).
• Check metal content and dependency.
• Check sensitivity against Cys and His reagents.

7

• Adenosylhomocysteinase

8

• Adenosylhomocysteinase
• Experimental results
• The enzyme has NAD as cofactor.
• When substrate is added, NADH is transiently
  formed.
• 2H2O exchanges at the 4 position of the
  substrate.
• 4-2H substrate shows kinetic isotope effect of
  1.5.
• 4,5-dehydroadenosine is processed to adenosine.
  

9

• Adenosylhomocysteinase, proposed mechanism

10

• Thermolysine
• Protease.
• What classes of proteases are there?
• How do you check which one thermolysine belongs
  to?

11

• Thermolysine
• Cbz-Gly-Leu-Leu is a substrate (cleaved after
  Gly).
• The phospoamidate is an inhibitor.

12

• Thermolysine
• L-Leucine hydroxamate and b-phenylpropionyl
  phenylalanine are inhibitors.

13

• Thermolysine
• The phosphoamidate could be an analogue of the
  transition state, the b-phenylpropionyl
  phenylalanine of a substrate, the L-leucine
  hydroxamate of a substrate with the attacking
  water molecule.

14

• Thermolysine
• L-Leucine hydroxamate and b-phenylpropionyl
  phenylalanine are inhibitors.

Zn2
Zn2
15

• 1-Aminocylopropane-1-carboxylate synthase

16

• 1-Aminocylopropane-1-carboxylate synthase
• E.C. 4.4.1.14
• Cofactor PLP
• Lys278 is completely conserved.
• Tyr152Phe/Gly mutants are 1,7/lt0,01 active

17

• 1-Aminocylopropane-1-carboxylate synthase

Mechanism from Li et al. (J. Exp. Botany, 2005)