Determination of the Kinetic and Chemical Mechanism of a Unique Peptidoglycan Recycling Enzyme with Dual Hydrolase and Kinase Functionality: Anhydromuramic Acid Kinase, AnmK

• Main Author: Clancy, Kathleen
• Other Authors: McCafferty, Dewey G
• Published: 2012
• Subjects: Chemistry; Biochemistry
• Online Access: http://hdl.handle.net/10161/5820

<p>Antibiotic  resistance  is  a  crisis  in  modern  society  causing  increasing  rates  of   bacterial  infections  impervious  to  current  therapies.    To  this  end,  new  targets  for   antimicrobial  treatment  must  be  pursued.    Peptidoglycan  recycling  is  an  understudied   key  life  process  in  the  bacterial  cell  where  over  60%  of  cell  wall  materials  are  reused   during  each  turnover.    Anhydro-­N-acetylmuramic  acid  kinase,  AnmK  is  a  novel  enzyme   in  this  pathway  catalyzing  the  conversion  of  anhydro-­N-acetylmuramic  acid  to  N-acetylacetylmuramic  acid-­6-­phosphate  in  the  presence  of  magnesium  and  ATP.    Previously,   several  crystal  structures  of  AnmK  have  been  solved  providing  insights  into  the  catalytic   mechanism,  but  until  this  point,  no  extensive  work  has  been  done.    The  goal  of  this  work   is  to  determine  the  chemical  and  kinetic  mechanisms  of  AnmK.    This  will  be  completed   using  a  continuous  assay  for  dual  hydrolysis  and  phosphorylation  activity  as  well  as  a   novel  assay  for  carbohydrate  hydrolysis.    Substrate  interactions  will  be  probed  using   previous  crystal  structures  as  a  guide.    Finally,  pre-steady-­state  studies  will  conclude  the   mechanistic  studies  giving  a  full  depiction  of  AnmK  catalysis.  </p><p>The  kinetic  and  chemical  mechanisms  of  AnmK  are  studied  in  the  steady-­state   using  a  continuous  assay  format  where  bisubstrate  kinetics  as  well  as  inhibitor  studies   were  performed  as  well  as  substrate  specificity  studies.    pH  rate  profiles  and  solvent   isotope  exchange  were  used  to  determine  the  residues  involved  in  catalysis.      </p><p>The  concerted  or  stepwise  nature  of  hydrolysis  and  phosphorylation  is  a  main   question  of  this  work.    A  novel  assay  using  glucose  oxidase  was  executed  to  trap  any   chemical  intermediates  formed  in  a  potential  stepwise  reaction.    This  assay  was  used  on wild-type  AnmK  as  well  as  a  variety  of  mutants.    Through  these  experiments,  two  key   residues  in  phosphoryl  transfer  are  identified  and  used  to  partially  decouple  hydrolysis   and  phosphorylation.  </p><p>Mechanistic  studies  were  continued  by  investigating  the  pre-­steady-­state  kinetics   of  AnmK  using  a  quench  flow  apparatus.    Wild-type  AnmK  showed  no  appearance  of  a   chemical  intermediate  during  timepoints  as  short  as  10  ms  and  also  showed  a  linear   formation  of  product  with  a  catalytic  rate  analogous  to  the  steady-­state  rate.    These   results  indicate  that  AnmK  undergoes  a  concerted,  one-step  catalytic  mechanism  with   no  chemical  intermediate.    AnmK  E330A  formed  both  hydrolysis  product,  as  well  as   hydrolysis  and  phosphorylation  product  in  the  pre-steady-­state  agreeing  with  the   previous  results  that  hydrolysis  and  phosphorylation  had  been  partially  decoupled.  </p><p>These  results  show  the  chemical  and  kinetic  mechanism  of  a  novel  enzyme  with   previously  undocumented  concomitant  hydrolysis  and  phosphorylation.    This  work   provides  further  understanding  of  carbohydrate-modifying  enzymes  as  well  as  the   peptidoglycan  recycling  pathway.    A  better  understanding  of  peptidoglycan   biosynthesis  and  recycling  could  lead  to  novel  antimicrobial  therapies  in  the  future.</p> === Dissertation