Mutations at the S1 Sites of Methionine Aminopeptidases from Escherichia coli and Homo sapiens Reveal the Residues Critical for Substrate Specificity

Abstract

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), a the two type I is that mutation of in in HsMetAP1 the S1 and with as the of and in S1 for the residues by hydrolysis of and by at the for MetAP for Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), a the two type I is that mutation of in in HsMetAP1 the S1 and with as the of and in S1 for the residues by hydrolysis of and by at the for MetAP for of the methionine from newly synthesized is and is by methionine aminopeptidase methionine EcMetAP1, coli MetAP type HsMetAP1, MetAP type MetAP type high methionine EcMetAP1, coli MetAP type HsMetAP1, MetAP type MetAP type high is with Met and a of the Met is the of Met and a and the of Met is that MetAP for the Met natural amino acid residues Met at this in Met by MetAP MetAP from and with of and with the is as of at this S1 binding pocket by the of that human type II MetAP only a for a amino acid as the and the Met is Met is by of the amino acid that this the of by two to only type I type II type I and type II is for the type I MetAPs from Escherichia coli (EcMetAP1) and and the type II MetAPs from and human of the S1 and binding and the residues in to the of MetAPs with the from a from Met and Met methionine and in a at the of EcMetAP1, the S1 with the acid at the N is from the natural and with that the S1 is to a of is to that type I and type II MetAPs use two different sets of amino acid to form the S1 and the for the residue for that the S1 of is by and and the S1 in is by and of type I MetAPs from coli and human and that of in the of the seven residues the S1 in and in HsMetAP1 and a and coli for HsMetAP1 of to the of MetAP for the of as and of the seven S1 residues in and the corresponding residues in HsMetAP1 by Ala for the of thiopeptolide and S1 residues that for hydrolysis and of of the thiopeptolide were synthesized in this and of is were by and from and of and S1 of by from coli and as as S1 of were the for the and from the a were by and coli for and the S1 were as and by and as of the from a of the as a by and of HsMetAP1 and S1 of HsMetAP1 the as with as the HsMetAP1 the as the to for the HsMetAP1 and with the were by were coli for were as and by a as were from the by of the from a and each of the as a by with of a thiopeptolide as a with and of at is a and for MetAP activity the of by that the to a of the thiopeptolide to for out at a in a and a and to the and at of hydrolysis of the thiopeptolide activity as a in by and were from of the in the of to by the were by of the of to the of is the and is the of by and by at the and the were by to the hydrolysis of the were at in a and a for HsMetAP1 a for by the of of and the at for for of and of to the by at a of of and S1 residues in and from is for and HsMetAP1 to the type I MetAP and of the seven S1 residues in in HsMetAP1, is that HsMetAP1 a to that of We of and by each of the seven residues with Ala and seven of HsMetAP1 and were by each of the seven corresponding residues and with of the S1 binding as in the of with a methionine for for for for and for and were in coli as and to with high and were in coli as and with HsMetAP1 were from the by the of and HsMetAP1 for of with each S1 residue by Ala were the residues and by activity in Met-S-Gly-Phe. and activity Tyr-65 and Trp-221 each and with the of the Met inactivity that hydrophobic with the is for binding and hydrolysis of a is in the and to the although is a the of the with in the the and in the this the with the to form with the of the and the for activity of of EcMetAP1, HsMetAP1, and S1 in as by and and and and the the residues and by the and were as as the and and only a that and with that the residues of the pocket corresponding of HsMetAP1 were found that were with from Mutation of Phe-197 in the and mutation of Trp-352 in a in the that in is is to and is in HsMetAP1 and and as with HsMetAP1, in and activity in in contrast to the full activity by the at the S1 of MetAPs from two different is that for MetAP for for Met as by is the for a Met is by the S1 of Met is with a as a the is in in the and the of the pocket as well as that of is that hydrophobic and the of the S1 pocket to Met and the with the of the of the is a natural amino acid residue by HsMetAP1, for hydrolysis by the for the were the S1 hydrolysis of by and by and and in the two residues that hydrolysis of mutation to Ala and of the mutation of to Ala the hydrolysis of is and in the hydrolysis of Met-S-Gly-Phe. as as of this in is in HsMetAP1 and to the only the seven S1 that the of Met and is a at the in of this in and in to Ala a to the at the the of this is the of the pocket the to Ala of with a of Met methionine and that is of the of the the of with acid the this of the is amino acid residue with a at the and to a only by HsMetAP1 the S1 of and and and by and S1 of and the in the Met is with that of the the and were the of the of of by Ala to the removal of the to the S1 two to and of is that of and the in as a to the in that and at to that for that the by to although is for the of of a to the S1 for of of of S1 by of the to the to the thiopeptolide with a to We and and the S1 that activity the and with of by EcMetAP1, HsMetAP1, and of of hydrolysis of by and HsMetAP1 were and of hydrolysis of by and HsMetAP1 were and in a as for by and the and and HsMetAP1 and the is with the from the that the cleavage of a with Met as the to that by the and in that the is to the S1 pocket caused hydrolysis of by the and is substrate, is that in the is by a in the in to the at the and is a is for the from the is that by and at a to that of of the EcMetAP1, HsMetAP1, and that a mutation at in in HsMetAP1 to Ala the S1 to the of only for a thiopeptolide for a of and MetAP Met from and Met as a from the of Met to and HsMetAP1 for with by the Met only to as by at and to the Met to HsMetAP1 and the of hydrolysis of by EcMetAP1, HsMetAP1, and of at is in of at is in in a the of of of HsMetAP1 in the is as a from cleavage the S1 the to and to and binding to of binding well with the of the S1 pocket caused by the residue at from to to the hydrolysis of and the of to the is the of the binding of to and is in with the from and the S1 and to to the and the Met from MetAP a in for and in and MetAP as a for and type I and type II MetAPs and only the of in is and type II human the type I HsMetAP1 and a is in for only is type and is essential for essential by the of the MetAP in coli and is to for MetAP the human type I the of each residue at the in the of and and the cleavage of the in the is in a MetAP with the for type I and type II MetAPs the and of the natural in and in and as MetAP a type I and type II human MetAPs at MetAP as by of by of the in the type I only at a is to that type I the for residues the S1 type I MetAPs as and HsMetAP1 and a pocket that the of Met of amino with a in by and binding a of the thiopeptolide is a natural amino acid residue to a by HsMetAP1, the for Met as the Met and a natural amino with a of and from is that in in HsMetAP1 as a to a with a as that of from this Mutation of this to Ala for with a at the to the and and of and with a at the to the S1 and of and and in as with contrast to the inactivity of that in this the the activity the from to as as the to for this the of Trp-221 in the hydrolysis of a with Met as the residue and the of in at the S1 site. systematic mutation of S1 with thiopeptolide and as S1 the of each residue to and in is and only and amino acid residues at the of and a the of and in in and in were to is that the and were to Met from that to for and MetAPs in the with of We by high of a a of that and for HsMetAP1 We in the of binding the and the the for from this and of the MetAP the of MetAP as of the methionine from newly synthesized is and is by methionine aminopeptidase methionine EcMetAP1, coli MetAP type HsMetAP1, MetAP type MetAP type high methionine EcMetAP1, coli MetAP type HsMetAP1, MetAP type MetAP type high is with Met and a of the Met is the of Met and a and the of Met is that MetAP for the Met natural amino acid residues Met at this in Met by MetAP MetAP from and with of and with the is as of at this S1 binding pocket by the of that human type II MetAP only MetAP a for a amino acid as the and the Met is Met is by of the amino acid that this the of by MetAPs two to only type I type II type I and type II is for the type I MetAPs from Escherichia coli (EcMetAP1) and and the type II MetAPs from and human of the S1 and binding and the residues in to the of MetAPs with the from a from Met and Met methionine and in a at the of EcMetAP1, the S1 with the acid at the N is from the natural and with that the S1 is to a of is to that type I and type II MetAPs use two different sets of amino acid to form the S1 and the for the residue for that the S1 of is by and and the S1 in is by and of type I MetAPs from coli and human and that of in the of the seven residues the S1 in and in HsMetAP1 and We a and coli for HsMetAP1 of to the of MetAP for the of as and of the seven S1 residues in and the corresponding residues in HsMetAP1 by Ala for the of thiopeptolide and S1 residues that for hydrolysis and of of the thiopeptolide were synthesized in this and of is were by and from and of and S1 of by from coli and as as S1 of were the for the and from the a were by and coli for and the S1 were as and by and as of the from a of the as a by and of HsMetAP1 and S1 of HsMetAP1 the as with as the HsMetAP1 the as the to for the HsMetAP1 and with the were by were coli for were as and by a as were from the by of the from a and each of the as a by with of a thiopeptolide as a with and of at is a and for MetAP activity the of by that the to a of the thiopeptolide to for out at a in a and a and to the and at of hydrolysis of the thiopeptolide activity as a in by and were from of the in the of to by the were by of the of to the of is the and is the of by and by at the and the were by to the hydrolysis of the were at in a and a for HsMetAP1 a for by the of of and the at for for of and of to the by at a of of of the thiopeptolide were synthesized in this and of is were by and from and of and S1 of by from coli and as as S1 of were the for the and from the a were by and coli for and the S1 were as and by and as of the from a of the as a by and of HsMetAP1 and S1 of HsMetAP1 the as with as the HsMetAP1 the as the to for the HsMetAP1 and with the were by were coli for were as and by a as were from the by of the from a and each of the as a by with of a thiopeptolide as a with and of at is a and for MetAP activity the of by that the to a of the thiopeptolide to for out at a in a and a and to the and at of hydrolysis of the thiopeptolide activity as a in by and were from of the in the of to by the were by of the of to the of is the and is the of by and by at the and the were by to the hydrolysis of the were at in a and a for HsMetAP1 a for by the of of and the at for for of and of to the by at a of of and S1 residues in and from is for and HsMetAP1 to the type I MetAP and of the seven S1 residues in in HsMetAP1, is that HsMetAP1 a to that of We of and by each of the seven residues with Ala and seven of HsMetAP1 and were by each of the seven corresponding residues and with and were in coli as and to with high and were in coli as and with HsMetAP1 were from the by the of and HsMetAP1 for of with each S1 residue by Ala were the residues and by activity in Met-S-Gly-Phe. and activity Tyr-65 and Trp-221 each and with the of the Met inactivity that hydrophobic with the is for binding and hydrolysis of a is in the and to the although is a the of the with in the the and in the this the with the to form with the of the and the for activity of of EcMetAP1, HsMetAP1, and S1 in as by and and and and the the residues and by the and were as as the and and only a that and with that the residues of the pocket corresponding of HsMetAP1 were found that were with from Mutation of Phe-197 in the and mutation of Trp-352 in a in the that in is is to and is in HsMetAP1 and and as with HsMetAP1, in and activity in in contrast to the full activity by the at the S1 of MetAPs from two different is that for MetAP for for Met as by is the for a Met is by the S1 of Met is with a as a the is in in the and the of the pocket as well as that of is that hydrophobic and the of the S1 pocket to Met and the with the of the of the is a natural amino acid residue by HsMetAP1, for hydrolysis by the for the were the S1 hydrolysis of by and by and and in the two residues that hydrolysis of mutation to Ala and of the mutation of to Ala the hydrolysis of is and in the hydrolysis of Met-S-Gly-Phe. as as of this in is in HsMetAP1 and to the only the seven S1 that the of Met and is a at the in of this in and in to Ala a to the at the the of this is the of the pocket the to Ala of with a of Met methionine and that is of the of the the of with acid the this of the is amino acid residue with a at the and to a only by HsMetAP1 the S1 of and and and by and S1 of and the in the Met is with that of the the and were the of the of of by Ala to the removal of the to the S1 two to and of is that of and the in as a to the in that and at to that for that the by to although is for the of of a to the S1 for of of of S1 by of the to the to the thiopeptolide with a to We and and the S1 that activity the and with of by EcMetAP1, HsMetAP1, and of of hydrolysis of by and HsMetAP1 were and of hydrolysis of by and HsMetAP1 were and in a as for by and the and and HsMetAP1 and the is with the from the that the cleavage of a with Met as the to that by the and in that the is to the S1 pocket caused hydrolysis of by the and is substrate, is that in the is by a in the in to the at the and is a is for the from the is that by and at a to that of of the EcMetAP1, HsMetAP1, and that a mutation at in in HsMetAP1 to Ala the S1 to the of only for a thiopeptolide for a of and MetAP Met from and Met as a from the of Met to and HsMetAP1 for with by the Met only to as by at and to the Met to HsMetAP1 and the of hydrolysis of by EcMetAP1, HsMetAP1, and of at is in of at is in in a the of of of HsMetAP1 in the is as a from cleavage the S1 the to and to and binding to of binding well with the of the S1 pocket caused by the residue at from to to the hydrolysis of and the of to the is the of the binding of to and is in with the from and the S1 and to to the and of and S1 residues in and from is for and HsMetAP1 to the type I MetAP and of the seven S1 residues in in HsMetAP1, is that HsMetAP1 a to that of We of and by each of the seven residues with Ala and seven of HsMetAP1 and were by each of the seven corresponding residues and with and were in coli as and to with high and were in coli as and with HsMetAP1 were from the by the of and HsMetAP1 for of with each S1 residue by Ala were the residues and by activity in Met-S-Gly-Phe. and activity Tyr-65 and Trp-221 each and with the of the Met inactivity that hydrophobic with the is for binding and hydrolysis of a is in the and to the although is a the of the with in the the and in the this the with the to form with the of the and the for activity of the the residues and by the and were as as the and and only a that and with that the residues of the pocket corresponding of HsMetAP1 were found that were with from Mutation of Phe-197 in the and mutation of Trp-352 in a in the that in is is to and is in HsMetAP1 and and as with HsMetAP1, in and activity in in contrast to the full activity by the at the S1 of MetAPs from two different is that for MetAP for for Met as by is the for a Met is by the S1 of Met is with a as a the is in in the and the of the pocket as well as that of is that hydrophobic and the of the S1 pocket to Met and the with the of the Met-S-Gly-Phe. of the is a natural amino acid residue by HsMetAP1, for hydrolysis by the for the were the S1 hydrolysis of by and by and and in the two residues that hydrolysis of mutation to Ala and of the mutation of to Ala the hydrolysis of is and in the hydrolysis of Met-S-Gly-Phe. as as of this in is in HsMetAP1 and to the only the seven S1 that the of Met and is a at the in of this in and in to Ala a to the at the the of this is the of the pocket the to Ala of with a of Met methionine and that is of the of the the of with acid the this of the is amino acid residue with a at the and to a only by HsMetAP1 the S1 of by Ala to the removal of the to the S1 two to and of is that of and the in as a to the in that and at to that for that the by to although is for the of of a to the S1 for of of of S1 by of the to the to the thiopeptolide with a to We and and the S1 that activity the and with as for by and the and and HsMetAP1 and the is with the from the that the cleavage of a with Met as the to that by the and in that the is to the S1 pocket caused hydrolysis of by the and is substrate, is that in the is by a in the in to the at the and is a is for the from the is that by and at a to that of of the EcMetAP1, HsMetAP1, and that a mutation at in in HsMetAP1 to Ala the S1 to the of only for a thiopeptolide for a of and MetAP Met from and Met as a from the of Met to and HsMetAP1 for with by the Met only to as by at and to the Met to HsMetAP1 and the the of of of HsMetAP1 in the is as a from cleavage the S1 the to and to and binding to of binding well with the of the S1 pocket caused by the residue at from to to the hydrolysis of and the of to the is the of the binding of to and is in with the from and the S1 and to to the and the Met from MetAP a in for and in and MetAP as a for and type I and type II MetAPs and only the of in is and type II human the type I HsMetAP1 and a is in for only is type and is essential for essential by the of the MetAP in coli and is to for MetAP the human type I the of each residue at the in the of and and the cleavage of the in the is in a MetAP with the for type I and type II MetAPs the and of the natural in and in and as MetAP a type I and type II human MetAPs at MetAP as by of by of the in the type I only at a is to that type I the for residues the S1 type I MetAPs as and HsMetAP1 and a pocket that the of Met of amino with a in by and binding a of the thiopeptolide is a natural amino acid residue to a by HsMetAP1, the for Met as the Met and a natural amino with a of and from is that in in HsMetAP1 as a to a with a as that of from this Mutation of this to Ala for with a at the to the and and of and with a at the to the S1 and of and and in as with contrast to the inactivity of that in this the the activity the from to as as the to for this the of Trp-221 in the hydrolysis of a with Met as the residue and the of in at the S1 site. systematic mutation of S1 with thiopeptolide and as S1 the of each residue to and in is and only and amino acid residues at the of and a the of and in in and in were to is that the and were to Met from that to for and MetAPs in the with of We by high of a a of that and for HsMetAP1 We in the of binding the and the the for from this and of the MetAP the of MetAP as the Met from MetAP a in for and in and MetAP as a for and type I and type II MetAPs and only the of in is and type II human the type I HsMetAP1 and a is in for only is type and is essential for essential by the of the MetAP in coli and is to for MetAP the human type I the of each residue at the in the of and and the cleavage of the in the is in a MetAP with the for type I and type II MetAPs the and of the natural in and in and as MetAP a type I and type II human MetAPs at MetAP as by of by of the in the type I only at a is to that type I the for residues the S1 type I MetAPs as and HsMetAP1 and a pocket that the of Met of amino with a in by and binding a of the thiopeptolide is a natural amino acid residue to a by HsMetAP1, the for Met as the Met and a natural amino with a of and from is that in in HsMetAP1 as a to a with a as that of from this Mutation of this to Ala for with a at the to the and and of and with a at the to the S1 site. and of and and in as with contrast to the inactivity of that in this the the activity the from to as as the to for this the of Trp-221 in the hydrolysis of a with Met as the residue and the of in at the S1 site. systematic mutation of S1 with thiopeptolide and as S1 the of each residue to and in is and only and amino acid residues at the of and a the of and in in and in were to is that the and were to Met from that to for and MetAPs in the with of We by high of a a of that and for HsMetAP1 We in the of binding the and the the for from this and of the MetAP the of MetAP as