is it possible to accomplish this in one s/// command?

Yes, but it is hideously ugly, and you shouldn't do it this way (update... do it BrowserUk's way):

use warnings;
use strict;
while (my $line = <DATA>){
    chomp $line;
    $line =~ s/(.{5})(.{4})/$1 . $2 . ' ' . substr($2, 0, 1) . 'Y' . s
+ubstr($2, 2, 2)/e;
    print $line . "\n";
}

__DATA__
col1 col2 col3 col4 col5
1234 5678 9012 3456 7890
[download]

prints:

col1 col2 cYl2 col3 col4 col5
1234 5678 5Y78 9012 3456 7890
[download]