Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5.
Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-(3)H(2),2-(14)C]-, [2-R-(3)H,2-(14)C]-, and [2-S-(3)H,2-(14)C]glycine, we found that in the overall process H(Si) is removed. Incubation of the enzyme with [2-RS-(3)H(2),2-(14)C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same (3)H:(14)C ratio as the starting substrate. The primary isotope effect was studied using [2-(2)H(2)]glycine, and we found that the specificity constants, k(cat)/K(M), for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M(-1) s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.
Jamil
Farrukh
F
School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
Gardner
Qurra-Tul-Ann Afza
QA
Bashir
Qamar
Q
Rashid
Naeem
N
Akhtar
Muhammad
M
D000594:Amino Acid Oxidoreductases; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011522:Protons
D011522:Protons; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase; D010100:Oxygen; D005998:Glycine