Aminoethylcysteine can replace the function of the essential active site lysine of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.
The biodegradative 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii catalyzes the NAD(+)-dependent conversion of (S)-HMG-CoA to (R)-mevalonate. Crystallographic analysis of abortive ternary complexes of this enzyme revealed lysine 267 located at a position in the active site, suggesting that it might serve as the general acid/base for catalysis. Site-directed mutagenesis and subsequent chemical derivatization were therefore employed to investigate this active site lysine. Replacement of lysine 267 by alanine, histidine, or arginine resulted in mutant enzymes that lacked detectable activity. Lysine 267 was next replaced by the lysine analogues aminoethylcysteine and carboxyamidomethylcysteine. Using instead of the wild-type enzyme the fully active, cysteine-free mutant enzyme C156A/C296A, lysine 267 was first replaced by cysteine. Cysteine 267 of mutant enzyme C156A/C296A/K267C was then treated with bromoethylamine or iodoacetamide to insert aminoethylcysteine (AEC) or carboxyamidomethylcysteine at position 267. The carboxyamidomethylcysteine derivative was inactive, whereas mutant enzyme C156A/C296A/K267AEC exhibited high catalytic activity. That aminoethylcysteine, but not other basic amino acids, can replace the function of lysine 267 documents both the importance of this residue and the requirement for a precisely positioned positive charge at the active site of the enzyme.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
D A
DA
Bochar
L
L
Tabernero
C V
CV
Stauffacher
V W
VW
Rodwell
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007460:Iodoacetamide / Q000737:chemistry; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
C000204:S-2-aminoethyl cysteine; D006903:Hydroxymethylglutaryl CoA Reductases; D008239:Lysine; D003545:Cysteine; D000409:Alanine; D007460:Iodoacetamide